WO2014127805A1 - Diagnosis method of pathogen - Google Patents

Abstract

The present invention is a diagnosis method of pathogen for identifying pathogen(s) involved in developing infection wherein there are a plural of pathogens which may be involved, comprising: a body fluid collecting step (S20) for collecting part of body fluid being provided from a test body and containing DNA; a DNA extraction step (S30) for extracting DNA from the collected body fluid to obtain DNA extract; a DNA amplification reaction step (S40) for conducting DNA amplification reaction in micro flow channels being configured in a substrate containing at least the same number of micro flow channels as the number of pathogens or of groups of pathogens to be detected, in the micro flow channels the DNA extract obtained in said DNA extraction step, and a PCR reagent including thermostable DNA polymerase, buffer, deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with amplified DNA being loaded, and primers being introduced prior to DNA amplification reaction in each of said micro flow channels in a manner that one kind of primers is never introduced in two or more micro flow channels, corresponding to each of a plural pathogens and being for amplifying each of target DNAs having sequence distinguishing each of said pathogens; and a detecting step (S50) for detecting labeling agent which reacts with the produced DNA by the DNA amplification reaction, wherein the pathogen corresponding to a primer being loaded in the micro flow channel in which the labeling agent is detected in said detecting step is identified as a pathogen involved in developing infection (S60).

Description

DIAGNOSIS METHOD OF PATHOGEN

TECHNICAL FIELD

[0001]

The present invention is related to a diagnosis method of pathogen . More specifically, the invention is related to a diagnosis method of pathogen for identifying pathogen(s) involved in developing infection wherein there are a plural of pathogens which may be involved.

RELATED ART

[0002]

For treating the infection, it is generally necessary to identify the pathogen ( s ) i nvo 1 ved in developing the infection, and then to admini ster drugs such a s antibiotics for wiping out the pathogen ( s ) . For identifying the pathogen ( s ) , traditional culture method is widely empl oyed . Such culture method requires a high 1 eve 1 of technical knowledge and skill (JP 2011-512159 A: Patent Document 1) .

[0003]

Further , JP 2001-204470 A (Patent Document 2 ) proposes a method for researching if a microorganism is included in a sample collected from a test body without going through the culture method . Patent Document 2 discloses a method including conduct ing DNA ampl i fication in accordance with PGR (polymerase chain reaction) method, staining the amplified DNA with fluorescence agent such a s ethidium bromide , detecting DNA amplification by agarose electrophoresis, and thereby identifying a target DNA of the pathogen which causes infection by the following method : preparing a DNA chip on which a probe being specifically bound to the target DNA is immobi 1 i z ed ; and bring the sample solution in which the DNA ampl i fication is detected to contact with the DNA chip to hybr idi z e the amplified DNA with the probe , then identifying whi ch probe involves the hybridization .

[0004]

WO 2009/110473 ( Patent Document 3 ) discloses a technique for identifying pathogen ( s ) involved in developing infection by the following method : extracting DNA from a sample collected from a test body , amplifying the DNA, detecting if target DNA ( s ) of the pathogen ( s ) exists in the sample by use of probe ( s ) being spec ifically bound to the target DNA, quant ifying the target DNA which is detected, and then comparing the developing level a s predetermined.

SUMMARY OF THE INVENTION

[0005]

I n the culture method of Patent Document 1 , the cultural operation of the sample collected from the test body is conducted with the prediction of the pathogen ( s ) from the symptoms. Therefore, the cultivation for the pathogen with the low level of the occurrence frequency tends to be laid over. Further , it is difficult to cultivate a pathogen on which the cultivation method is not e s t abl i shed . Moreover , there is possible a pathogen whi ch may not be ident i f ied because of its biochemical characters , even if the cultivation is successful . Owing to the above circumstances, it is difficult to practice the appropriate treatment for the infection over the patient , since the immediate di agno s i s is failed to be made .

[0006]

I n the culture method, a microorganism is f i shed out of colonies appearing by cultivation, and then the f i shed microorganism is ident i f ied . Since the condition for identifying is di f fer ent from each of pathogens , di agno s i s is complicated . I n particular, for community-acquired pneumonia , the immediate di agno s i s is difficult . Moreover , for the infection caused by a plural of pathogens , it requires some amount of time to identify all of the pathogens . I n view of these circumstances, it is di f f i cul t to practice the

appropriate treatment for the infection over the patient .

[0007]

I n the techniques of Patent Document s 2 and 3 , it is necessary to take operations for identifying pathogen ( s ) i nvolved in deve loping infection separately, after the ampl i fication of DNA . I n view of reali z ing the immediate and simple diagnosis, those techniques are still insufficient.

[0008]

The present invention is accomplished in order to solve the above mentioned problems, and has intent to provide an immedi at e and simple di agno s i s technique for identifying pathogen ( s ) involved in developing infection, wherein there are a plural of pathogens which may be i nvo 1ved .

[0009]

Namely, the present invention is directed to the following :

(1) A di agno s i s method of pathogen for identifying pathogen ( s ) involved in developing infection wherein there are a plural of pathogens which may be involved, compr i sing :

A body fluid collecting step for collecting part of body fluid being provided from a test body and containing DNA ;

A DNA extraction step for extracting DNA from the collected body fluid to obtain DNA extract ;

A DNA ampl i fication reaction step for conducting DNA amp lification reaction in mi cr o flow channel s be i ng conf igur ed in a substrate containing at least the same number of mi cro f low channels as the number of pathogens or of groups of pathogens to be detected, in the micro flow channels the DNA extract obtained in said DNA extraction step, and a PGR reagent including thermostable DNA polymerase , buffer ,

deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with amplified DNA being loaded, and primers being introduced prior to DNA ampl i fication reaction in each of said mi cr o flow channels in a manner that one kind of primers is never introduced in two or more micro flow channel s , corresponding to each of a plural pathogens and being for amplifying each of target DNAs having sequence distinguishing each of said pathogens ; and

A detecting step for detecting a labeling agent which reacts with the produced DNA by the DNA amplification reaction, Wherein the pathogen corresponding to a primer being loaded in the micro flow channel in which the labeling agent is detected in said detecting step is identified as a pathogen involved in developing infection.

(2) The di agno s i s method a s spec i f i ed in (1), wherein said substrate further compri ses a micro flow channel for a positive control .

(3) The di agno s i s method a s spec i f i ed in (2), wherein said substrate further compri ses a micro flow channel for a negative control .

(4) The diagnosis method a s specified in any of (1) to (3), wherein a temperature control for the DNA ampl i fication reaction is conducted by heating by s team and cooling by dry air said substrate in said DNA amplification reaction step. (5) The diagnosis method a s specif i ed in any of (1) to (4), wherein said PGR reagent is introduced together with said DNA extraction reagent in extraction reaction conducted in said DNA extraction step.

[0010]

By virtue of the present invention, it becomes possible to identify pathogen ( s ) involved in causing infection immediately and easily. As a consequence, the appropriate treatment for the infection should be effectively practiced, so that it is possible to avoid the spread of the infection. BRIEF DESCRIPTION OF DRAWINGS

[0011]

FIG. 1 shows a diagram illustrating an embodiment of the substrate employed for the embodiment of the invention ;

FIG. 2 shows a diagram illustrating another embodiment of the substrate employed for the embodiment of the i nvent i on ;

FIG. 3 shows a diagram illustrating an embodiment of a substrate holder empl oyed for the embodiment of the invention ;

FIG. 4 sho s a cross-section view of a concrete embodiment for the substrate holder of FIG. 3 ;

FIG. 5 shows a cross-section view of an alternate for the substrate holder of FIG. 3 ;

FIG. 6 shows a diagram illustrating a function of means for sealing empl oyed for the embodiment of the invention ;

FIG. 7 shows a temperature profile in controlling temperature for the DNA amplification in the embodiment of the invention ;

FIG. 8 shows a diagram illustrating a thermal cycler in the embodiment of the invention;

FIG. 9 shows a principal diagram illustrating the thermal cycler of FIG. 8; and

FIG. 10 shows a f lowchar t illustrating the embodiment of the invention .

DETAILED DESCRIPTION

[0012]

The invention is described be 1 ow .

From one aspect , the present invention provide s a pathogen diagnosis system for identifying pathogen (s) involved in developing infection wherein there are a plural of pathogens which may be i nvo 1ved .

The pathogen diagno s i s system compr i s e s a body fluid collecting means for collecting part of body fluid be i ng provided from a test body and containing DNA ; a DNA extraction reagent for extracting DNA from the collected body fluid by the body fluid collecting means to obtain DNA extract ; a PGR reagent for amplification reaction of DNA on the DNA extract , compr i sing thermostable DNA polymerase , buf f er ,

deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the ampl i f i ed DNA ; a plural types of primer reagent s each compr i sing one of more kinds of primers for amplification of a target DNA having sequence distinguishing each of a plural of pathogens, each primer corresponding to each of the pathogens; a DNA extraction container for providing a field for DNA extraction reaction by use of the DNA extraction reagent ; an extract collecting means for collecting part of DNA extract obtained in the DNA extraction container; a substrate containing at least the same number of mi cro flow channel s a s the number of pathogens or of groups of pathogens to be detected, in the micro flow channels the DNA extract collected by the extract collecting means , the PGR reagent and the primer reagent being loaded, and a field for DNA ampl i fication reaction be i ng given ; and a thermal cycler for controlling the temperature for DNA ampl i fication reaction in the micro f low channel by heating or cooling the substrate .

[0013]

A body fluid collecting means is a piece of equipment for collecting body fluid from a patient who is a test body , and includes a cotton-tipped applicator , a dropper , a pipette and the like. Among these, a cotton-tipped applicator is suitable in view of easy-to-use of handl ing . When it is difficult to directly collect a sample , collection may be conducted first by taking up the body fluid by use of something to hold the body fluid, such a s petr i dish and filter paper , and then by collecting a sample therefrom by use of a cot ton-tipped applicator, a dropper, a pipette and the like. Further, in holding the body fluid, it is preferable to remove the moisture of the fluid, since it becomes easier to take up a sample. Specifically, a filter paper is employed as an element to take up a body fluid, and then f o lded with placing the body fluid i nward so that the moi sture is allowed to be absorbed, and then unfolded and subjected to sampling by use of an element to collect the dehydrated body fluid, such as a cotton-tipped applicator, a dropper, a pipette and the like. Such a combination of those elements in order to realize the collection of body fluid is also included by the scope of the body fluid collecting means.

[0014]

For the embodiment of the invention, each of reagents, such as a DNA extraction reagent for extracting DNA from the body fluid, and a PGR reagent for amplification reaction of DNA, and primer reagent s .

[0015]

The DNA extraction reagent contains enzyme ( s ) for destructuring the cell walls of the cell contained in the body fluid being collected from the test body , and enzyme ( s ) for breaking up proteins contained in the body fluid, and en z yme ( s ) for breaking up mucus contained in the body fluid, and also contains a component for changing a form of DNA existing in the cell so that DNA is allowed to be easily extracted, such as a surfactant including SDS (sodium dodecyl sulfate) . Further, the commercially available DNA extraction reagent may be suitably empl oyed , in particular , two -component type to allow the reaction to proceed in two stages is preferable, in view of handl i ng .

[0016]

The PGR reagent contains thermostable DNA polymerase, buffer, deoxynucleoside triphosphates (dNTP), and labeling agent being spectroscopically detectable by reacting with the ampl i f i ed DNA. The thermostable DNA polymerase includes a DNA polymerase suitable for ordinary PGR reaction, such as a DNA polymerase derived from thermus aquat i cu s ( US Patents

4,889,818 and 5,079,352) (Trade name: Taq Polymerase), a DNA polymerase derived from Thermus thermophilus (WO 91/09950) ( rTth DNA polymerase ) , a DNA polymerase derived from Pyrococcus fur iosus (WO 92/9689) (Pfu DNA polymerase ; manufactured by Stratagenes Corp. ) , a DNA polymerase derived from Thermococcus litoralis ( EP-A- 455430) (Trade mark : Vent ; New England Biolabs Inc. ) . Further , for example , a polymerase being def i ci ent in 5 ' to 3 ' exonuclease activity is preferable . By u s i ng such a polymerase being deficient in 5 ' to 3 ' exonuclease activity, it could be avoided to cleave a primer by a polymerase . Such a polymerase being de f i ci ent in 5 ' to 3 ' exonuclease activity as commercially avail able includes for example Gene Taq (NIPPON GENE CO . , LTD . ) , TITANIUM Taq ( Clontech Laboratories Inc . ) , ATth (TOYOBO CO., LTD.) .

[0017]

Moreover, buffers and dNTPs include ordinary products for PGR reaction . For example, dNTPs include dATP, dCTP, dGTP, dTTP and dUTP , and the mixture thereof is suitably employed . For example, buf f er s include a solution exhibiting a suitable pH for the DNA ampl i fication by PGR method, such a s Tris-HCl, Tricine, ME S , MOPS , HEPES and CAPS . Furthermore , labeling agent includes a compound being capable of labeling DNA by reacting with DNA . Typi cal examples include fluorescence agent emitting fluorescence by binding to the ampl i f i ed DNA, such a s SYBR(tm) GREEN and ethidium bromide .

[0018]

Further , the PGR reagent may contain solvent such as water and buf f er , glycerol , heparin, betaine, KC1, MgC 1₂ and MgS0₄.

[0019]

The primer reagent contains one or more kinds of pr imer s , each of whi ch is employed for amplifying target DNAs , respectively, each having a di s t inc t i ve sequence for the corresponding pathogens to be detected . That is, in the case that the pathogen (s) to be detected exist in the body fluid from the test body , the primer ( s ) is to initiate the DNA ampl i fication reaction by PGR method by use of the extracted DNA ( s ) derived from pathogen ( s ) a s a template . Since the target for the diagnosis is the infection wherein there are a plural of pathogens which may be involved, the primer reagents should be separately prepared for every primer corresponding to each of these pathogens. Alternatively, the primer reagent may be a group of primers, each primer being employed for the ampl i fication of DNAs , correspond to a plural of pathogens which does not influence on one another. It should be noted that each of groups contains the different primers from one another .

[0020]

A DNA extraction container has a certain amount of volume , and provides a field for DNA extraction reaction of the body fluid collected by the body fluid collecting means by use of the DNA extraction reagent . The DNA extraction container is suitable in a form of a plastic tube , for example a mi cr o tube , in view of handling . The tube is preferred to be siliconized on the inner surface .

[0021]

11 is po s s ible to conduct the extraction reaction in the DNA extraction container . However, it is preferable to appropriately regulate the temperature of the reaction in view of reaction efficiency.

For example, the following temperature regulation may be effective : the reaction mixture is first heated to around 70 degrees C, to thereby activate enzymes included in the DNA extraction reagent , and then the reaction is initiated ; and the reaction mixture is allowed to keep that condition for 6 minutes or so, to thereby progress the extraction reaction; then , the temperature of the reaction mixture is e 1 evat ed to 94 degrees C, to thereby inactivate the enzymes and to terminate the reaction .

Such a temperature regulation may be easily realized by use of a temperature regulator for regulating the temperature for initiating or stopping the extraction reaction in the DNA extraction container , such a s heating blocks .

[ 0022 ]

The PGR reagent may be introduced into the extraction reaction resultant by the DNA extraction reaction (hereinafter referred to " DNA extract") . However , it is preferable to introduce the PGR reagent into the DNA extraction container together with the DNA extraction reagent in view of workability, since the activating temperature of the enzymes of the DNA extraction reagent is different from that of the PGR reagent .

[ 0023 ]

An extract collecting means is a piece of equipment for collecting part of the DNA extract in the DNA extraction container, including for example a dropper and a pipette in the case of collecting a s an extract the supernatant of the solution, and a tube with a membrane filter in the case of removing the other substances than the extract . When empl oyi ng the tube , it may be combined with the DNA extraction container . [0024]

A substrate contains a feed for introducing the DNA extract; and a plural of micro flow channels, each being configured in a manner that one end is connected with the feed for introducing the DNA extract , and that the other end is open .

Further , the substrate is made from a material which is unreactive with any of the DNA extraction reagent, the PGR reagent nor the primer reagent, preferably quartz in view of its excellent thermal conduct ivity . Moreover , there is no limit in the configuration .

[0025]

One of concrete embodiments is described, for example, in WO 2012/ 001972. That IS, 3. S shown in FIG. 1 , the substrate 40 is made up in a circle form and has a hole at the center which becomes a feed 11 for the DNA extract . Further , eight of mi cr o flow channel s 21a, 21b, 21c, 2 Id, 21 e , 21 f , 21 g and 2 Ih are formed in the s ame length to radiate from the center part of the substrate 40 , in each of whi ch one end is connected with the feed 11 , and the other end, that is, the end at side of the outer circumference of the substrate 40 , is open .

[ 0026 ]

Further , the substrate 40 has the discoid shape with about 10 mm in diameter and about 0.3 mm in thickness. The cross-section surface of the micro f low channel s is 100 μιη s quare in size. By conf igur ing the substrate and the mi cro f low channel s in such form and size, it is r eal i zed to cut down the volume of the test solution to some microliters in total for all micro flow channels, so that heat content required for the test solution can be significantly reduced .

[0027]

For the procedure in loading the pr imer s in the substrate 40, the procedure is conducted for each of micro f low channels 21a to 21 h , separately : first, a primer reagent is dropped at the opposite end of the feed 11 by use of a dropper and the like and introduced into the channel by the capillarity effect, so that the different primer reagent is introduced into each of the mi cr o f low channels, respecti ely. Thereafter, the substrate is sub ected to drying, so that a primer is loaded in each of mi cro f low channels .

[0028]

A DNA extract is introduced into the micro f low channels from the feed 11 by the capillarity effect . That is, when the DNA extract is brought into contact with the feed 11 , the extract actively f lows in the direction indicated by arrows of FIG. 1 in each of the micro f low channel s 21a, 21b, 21c, 2 Id, 21 e , 21 f , 21 g and 21 h by the capillarity effect, so that each of the mi cro f low channel s 21a, 21b, 21c, 21 d , 21 e , 21 f , 21 g and 2 Ih will be filled surely with the DNA extract by its surface tension effected at the open end of each of the mi cro f low channel s . [0029]

I n FIG. 1 , it is illustrated that the substrate is conf igured in a circle form. However, a s shown in FIG. 2 , the substrate may be configured in a rectangular or square form.

I n FIG. 2 , from planer view of the substrate 41 , the substrate 41 has the mi cro f 1 ow channels 22a to 22s and 23a to 23s which are formed in parallel to one another in the direction of one s ide and arranged in the direction of the other side at certain interval , such a s 0.5 mm . I n the substrate 41, at the s ide of one end of each of micro f low channels a feed lib for the DNA extract , which is conf igured in a window form in the direction of lining up the mi cro flow channel s in planar view of the substrate 41. Further , on the other side of the micro flow channel s , a series of primer feeds 11a is formed for introducing the primer reagents . The number of the mi cro flow channels is at least the s ame number of pathogens to be detected or groups of pathogens to be detected .

[0030]

Further , for the procedure in loading the primers in the substrate 41 , the procedure is conducted for each of micro flow channel s 22a to 22s, separately : first, a primer reagent is dropped at one of the primer feed 11a by use of a dropper and the like and introduced into the channel by the capillarity effect, so that the different primer reagent is introduced into each of the micro flow channels, respectively. Thereafter, the substrate is subjected to drying, so that a primer is loaded in each of micro flow channels. A DNA extract is introduced into the mi cro flow channel s from the feed lib by the capillarity effect . I n FIG. 2 , although there is not a window like the primer feeds 11a at the opposite side of the feed lib of each of the mi cro flow channels 23a to 23s, those micro flow channels 23a to 23s may be used as negat i ve control ( s ) a s mentioned below. I f the micro flow channels 23a to 23s are also to be used a s a space for detecting the DNA amplification, a window like the primer feed 11a may be configured at the opposite side of the feed lib of each of the mi cro flow channels 23a to 23s.

[0031]

Prior to the DNA amplification, a primer is introduced in each of micro flow channel s in the substrate . 11 is needed to make sure that one kind of primers is never introduced in two or more micro flow channels that is, each of the mi cro flow channel s contains the different primer ( s ) from one another I n particular, when one kind of a primer is in one mi cro flow channel , it means that one sole different primer is introduced in each of channel s . Even when a group of a plural of primers is in one mi cro flow channel , it is needed to make sure that one kind of primers is never included in two or more groups, so that the s ame primer never exists in two or more mi cro flow channel s .

[ 0032 ] According to such a configuration, in the micro flow channels, the PGR reagent and the primer (s) are loaded in advance; once the extraction reaction resultant being collected by the extract collecting means is introduced, a field for the DNA amplification reaction is provided under the prescribed condition of heating by the temperature control for the DNA amplification reaction as mentioned below.

[ 0033 ]

Further, it is preferable to arrange a micro flow channel for a positive control .

Her e , the term "a positive control" means a control for representing a state that DNA ampl i fication reaction takes place , so that the criteria for evaluating whether DNA ampl i fication reaction takes place or not in a micro flow channel , whi ch is treated for DNA ampl i fication reaction of the target DNA, may be provided . That is, by comparing the micro flow channel of the target DNA with the positive control in the detection level ( as mentioned be 1 ow ) , it may be evaluated whether the DNA amplification reaction takes place or not in the mi cro flow channel of the target DNA .

Therefore, to the micro flow channel of the positive control are introduced in advance a template DNA (hereinafter referred to "standard t emp late") having the different sequence from any of the target DNAs and a primer ( hereinafter referred to " standard primer" ) having the sequence compl ement ary to a part of the sequence of the standard template. Here, the term "the detection level" means a spectroscopic intensity of the 1 abe 1 i ng agent ; the higher detection level shows "positive", which means that the labeling agent is detected in a higher amount : and the lower detection level shows "negative", which means that the labeling agent is detected in a smaller amount .

According to such a configuration, to the micro f low channel of the positive control is introduced the DNA extract and the temperature control as mentioned be 1 ow is conducted, so that the DNA ampl i fication reaction takes place between the standard primer and the s t andard template . I n other words , if a DNA ampl i fication reaction does not take place in the positive control , it may be evaluated that the reaction condition such a s a temperature cycle is not appropriate .

[0034]

Further , it is preferable to arrange a mi cr o f low channel for a negative control .

Her e , the term " a negative control" means a control for i ndi cat i ng a detection level ( a s mentioned above ) of the substrate itself in detecting DNA after treatment of DNA ampl i fication reaction, so that the background intensity of the substrate is provided . That is, particularly when the DNA detection level is low, by compar i ng the micro flow channel of the target DNA with the negative control , it may be evaluated whether the detection level of each of micro f low channel indicates the amplified DNA or the background of the substrate.

Therefore, no primer is introduced to the micro flow channel of the negative control. According to such a configuration, even when the DNA extract is introduced to the micro f low channel of the negative control , and the temperature control as mentioned below is conducted, no DNA amplification takes place in the micro flow channel of the negative control.

[0035]

Furthermore, it is preferred to provide a sealing means for sealing the open end of each of the mi cro f low channel s , so that the DNA extract introduced thereinto may be prevented from the evaporation at the open end of the micro flow channel. Such sealing means may include any material to seal the open end, preferably a substance in a 1 iquid form in view of wor kabi 1 i t y and the like. I n particular, a 1 i qu i d having high boiling point is preferable , since it does not vaporize under the heating condition in the DNA amplification reaction. Mo s t preferable is mineral oil, in view of handl i ng .

[ 0036 ]

Here is illustrated a holder for holding the substrate a s an embodiment for employing the sealing means . FIG. 3 shows one embodiment of the holder . Fig . 4 shows a cross-section diagram of the holder .

[0037]

I n F I Gs 3 and 4, the substrate holder 50 is conf igured by an upper part 50a and a lower part 50b. The upper part 50a is provided with a guide 51a penetrating in a tapered shape through the upper part 50a for introducing the DNA extract into the substrate 40, and a sealing material feed 52. The lower part 50b is provided with an opening 51b cylindrically penetrating through the lower part 50b. Further , between the guide 51a and the opening 51b, a holding part is provided for holding the substrate 40. Moreover , when the upper part 50a and the lower part 50b are brought into contact , an open space 53 for the sealing material appear s ad acent to the holding par t , so that the open space 53 for the sealing material has a contact with the outer circumference of the substrate 40 when holding the substrate 40. The sealing material feed 52 penetrates through the upper part 50a and is communicated with the open space 53.

[0038]

According to such a configuration, by separating the upper part 50a and the lower part 50b, holding the substrate 40 at the holding part of the substrate holder 50 in whi ch the feed 11 is faced toward the upper part 50a, and bringing into contact with the upper part 50a and the lower part 50b again, the substrate 40 is held in the substrate holder 50. Further , by dropping the DNA extract collected by the extract collecting means down to an opening of the guide 51a, the DNA extract is introduced into the mi cr o f 1 ow channel s 21a to 2 Ih through the feed 11 on the substrate 40. Moreover, mineral oil is introduced as a sealing means through the sealing material feed 52 into the open space 53 for the sealing material, and flows around the outer circumference of the substrate 40, and into each of the micro flow channel s .

[0039]

In FIG. 4, it is illustrated that the shape of the opening 51b provided on the lower part 50b is in a cylindrical form. However, as shown in FIG. 5 , the lower part 50c may be employed which is provided with the opening 51c of a tapered shape . By conf igur ing the opening 51c in such form, it is hard to retain vapor and air on the opening, so that it is realized to control the temperature for the DNA amplification reaction more accurately .

Further , in F I Gs . 4 and 5 , the guide 51a may be formed in a cylindrical form . However, by conf igur ing it in a tapered form a s shown in F I Gs . 4 and 5 , there is conveniently no shade in making an observation on the micro f 1 ow channel s after the DNA ampl i fication reaction is conducted in the substrate 40.

[0040]

I n FIG. 6 , the function of the sealing means is illustrated with the enlarged principal diagram of one micro flow channel and the open space 53 for the sealing material in vicinity thereof .

According to FIG. 6 , the mi cr o flow channel 21a is filled with the DNA extract 31 in the substrate 40 being held by the substrate holder 50. On the other hand, the open space 53 for the sealing mat er i al has a contact with the end of the mi cro f low channel 21a. Mineral oil 33 being introduced as the sealing means f lows in the open space 53 for the sealing material , and then reaches to the outer circumference of the substrate 40. At this time , the mineral oil 33 flows in the micro f 1 ow channel 21a from the open end thereof . Accordingly, the open end of the micro f 1 ow channel 21a is sealed by the mineral oil 33 , resulting in the air layer 32 next to the mineral oil 33 in the mi cro f low channel 21a, so that the DNA extract may be pre ented from the evaporation by heating at time of the DNA ampl i fication reaction .

[0041]

A thermal cycler controls the heating condition for the

DNA ampl i fication reaction in the micro f low channel s of the substrate . A thermal cycle in accordance with a temperature profile a s shown in FIG. 7 may be exempl i f i ed as the heating condition .

I n FIG. 7 , the temperature profile is represented by the relationship of heating temperature to the e 1 ap s ed time . Spec ifically, the temperature profile includes a preheating period from room temperature ( RT ) to annealing temperature ( T_L ) ( t_i to t₀); an initial heating period from the temperature T_L to denaturing t emperatur e T_H ( 1₀ to 11 ) ; a denaturing period at the temperature T_H ( 11 to t₂) ; a cooling period from the temperature T_H to the temperature T_L (t₂ to t₃) ; an annealing period at the temperature T_L ( t₃ to t₄) ; a first heating period from the temperature T_L to DNA melting temperature (T_M) (t₄ to 15 ) ; an e 1 ongat i on reaction period at the temperature T„ (t₅ to 1₆ ) ; and a second heating period from the temperature T_M to the temperature T_H (t₆ to t₇) . The thermal cycle of 11 to t - is repeated by prescribed times.

[0042]

For example, in the thermal cycler , the temperature T_L is set to a temperature from 53 degr ee s C to ( 63 ± 0.5) degrees C; and the temperature T_M is set to a temperature from 70 to 72 degrees C; and the temperature of T_H is set to a temperature from 95 to 99 degrees C . Further , the period of the time is set a s follows : t_i to 1₀ , 5 seconds or less 1₀ to 11 , 0.5 second or less; 11 to 1₂ , 0.2 second or less; t₂ to t₃ , 0.5 second or less; 13 to 1₄ , 0.2 second or less; t₄ to 1₅ , 1 second or less; 15 to 1₆ , 2.5 seconds or less; t₆ to 1₇ , 0.5 second or less. Moreover , the one total temperature cycle ( 11 to 1₇ ) is set to 5 seconds or less. Furthermore , the temperature cycle is repeated by at least 20 times, and preferably about 35 times.

[0043]

For realizing such temperature control for the DNA amplification reaction, a steam thermal cycler (heating and cooling apparatus ) is suitably employed, which is conf igur ed in a manner to control the temperature for the DNA amplification reaction by heating by steam and cooling by dry air the substrate .

[0044]

FIG. 8 shows a diagram illustrating the configuration of a steam heating and cooling apparatus as a thermal cycler in the embodiment of the invention . FIG. 9 shows a principal diagram illustrating the heating and cooling apparatus of FIG. 8.

According to FIGs . 8 and 9 , the heating and cooling apparatus 20 contains a motor 60 equipped with an axi s 60a carrying a supporting board 60b for supporting the substrate 40 and being capable of rotating the supporting board 60b in the direction of the arrow R ; a steam nozzle 61 conf igured a s facing to the rotating tra ectory of the substrate 40 by action of the motor 60 ; a steam generator 71 for generating and feeding steam to the s team nozzle 61 ; an air noz z le 62 configured as facing to the rotating tra ectory of the substrate 40 by action of the motor 60 ; a blower 72 for generating and feeding air having a lower temperature than the s team from the steam noz z le 61 to the air nozzle 62 ; an air nozzle 63 conf igured a s facing to the rotating trajectory of the substrate 40 by action of the motor 60 ; a blower 73 for generating and feeding air having a lower temperature than the air from the air noz z le 62 to the air nozzle 63 ; a control unit 70 for controlling the action of the motor 60, the steam generator 71, the blower 72 and the blower 73. Further , not shown in F I Gs , the heating and cooling apparatus 20 is provided with a water supplier for supplying water for the steam to the steam generator 71.

[0045]

I n this embodiment , a steam f low of the saturated water vapor is empl oyed as the airflow at high temperature , and a dry air is employed a s the air f low at lower temperature than the steam and the ai r f low at further lower temperature . However , there is no limitation, a s long a s the substrate 40 may be heated or cooled at the prescribed temperature ; that is, when pass i ng over the steam nozzle 61 , the substrate 40 is heated to the temperature T_H ; and when passing over the air nozzle 62 , the substrate 40 is heated ( or cooled ) to the temperature T_M ; and when pass i ng over the air nozzle 63 , the substrate 40 is cooled to the temperature T_L.

[0046]

The control unit 70 is suppl ied with electricity by an outer electrical source not shown in FIG. The steam generator 71 , and two of the blowers 72 and 73 is electrically connected with the control unit 70 , to thereby be suppl ied with the electricity and controlled .

[0047]

Here is i 1 lu s t rat ed the heating and cooling procedure of the substrate 40 by the heating and cooling apparatus 20 in accordance with the temperature profile as shown in FIG. 7.

[0048]

(1) Attaching and supporting the substrate 40 on the end of rotating side of the supporting board 60b;

(2) Starting the steam generator 71 , and two of the blowers 72 and 72 by action of the control unit 70 , so that the substrate 40 is ready for subjecting to heating or cooling at the preset temperature when passing over the corresponding nozzles ;

(3) Starting the motor 60 by action of the control unit

70 , so that the substrate 40 supported by the supporting board 60b is mo ed over the air nozzle 63 to thereby be sub ected to the preheating period (t_i to 1₀ ) , and thereafter moved over the steam nozzle 61 to thereby be sub ected to initial heating period ( 1₀ to ti ) , and maintained at that temperature for prescribed time ( 11 to 12 ) , so that the DNA denaturing occurs in the mi cr o flow channels of the substrate 40 ;

(4) Moving the substrate 40 , passing over the air nozzle 62 to the upper place to the air nozzle 63 to thereby be sub j ected to the cooling period ( 1₂ to 1₃ ) , and maintained at that temperature for prescribed time ( 1₃ to t₄) , so that the annealing occurs in the mi cro flow channels of the substrate 40 ;

(5) Further moving the substrate 40 over the air nozzle 62 to thereby be sub ected to the first heating period (t₄ to 15 ) , and maintained at that temperature for prescribed time (t₅ to 16 ) , so that the DNA amplification (elongation) reaction occurs in the mi cro f low channels of the substrate 40;

(6) Further mo ing the substrate 40 over the stream nozzle 61 to thereby be sub ected to the second heating period (t₆ to 17 ) , so that one cycle of the temperature control is over ;

(7) Repeating the temperature control shown as the temperature profile of 11 to 1₇ , the above steps (3) to (6) for prescribed time s , and then finally br i ngi ng the substrate 40 back to the position a s shown in F I Gs 8 and 9 , heating and cooling procedure is terminated .

[0049]

I n the above step ( 3 ) , right after steam fed by the steam nozzle 61 reaches to the surface of the substrate 40 , the s team become s concentrated to be minuscule droplet s of water, so that by the droplet s of the high temperature the substrate 40 whi ch has the excellent thermal conductivity is rapidly heated from the temperature T_L to the temperature T_H , and maintained .

[ 0050 ]

I n the above step (4), when feeding the air from the air nozzle 63 to the substrate 40 , the concentrated and minuscule droplet s of water evaporate to thereby cool the substrate 40 from the temperature T_H to the temperature T_L, and maintained . I n the cooling process by the dry air , the minuscule droplet s of water can be rapidly evaporated, the substrate 40 can be cooled down quickly . [ 0051 ]

In the temperature control for the DNA amplification reaction, the temperature of the substrate 40 may be measured by a thermometric means such as a thermocouple to thereby change appropriately the heating or cooling condition at each of steps in accordance with the measured temperature , such as adjusting the steam temperature generated by the steam generator 71, and adjusting the air temperature generated by the blowers 72 and 73, and adjusting time for maintaining the substrate 40 above each of the nozzles .

[ 0052 ]

The conventional heating and cooling apparatus requires a continuous power supply , so that an external power supplier is general ly used. On the other hand, according to the present invention, the heating or cooling time should be shorter, so that a battery or a portable second cell may be employed as a power source . Therefore, the heating and cooling apparatus could be portable , so that the apparatus could be used in other place than laboratory, in particular, out s ide . Further , the substrate is made from quar t z , so that the thermal conductivity is further improved, and then such an effect should increase .

[ 0053 ]

Further , the pathogen di agno s i s system may contain a spectrometer for spectroscopically detecting 1 abe 1 i ng agent which reacts with the produced DNA by the DNA ampl i fication reaction. Such spectrometer makes it possible to detect the DNA amplification reaction, even in the case of showing the spectroscopically low sensitivity because of shorting the DNA content in the body fluid .

[0054]

Since the DNA ampl i fication reaction is detected in the micro flow channel means that the primer which is allowed to be introduced therein in advance should involve the DNA ampl i fication reaction, so that it is sugge s ted that the DNA being r eact i ve with the primer a s a template DNA is included in the DNA extract from the body fluid . Therefore, it is recognized that a person providing the body fluid develops the infection involved by the pathogen ( s ) which should cor r e spond to the primer ( s ) causing the DNA amplification reaction . Accordingly, in the case that the DNA amplification reaction is detected, identifying the micro flow channel ( s ) and the primer ( s ) introduced therein makes it possible to identify the pathogen ( s ) which deve 1 op s the infection in a person providing body fluid .

[0055]

From another aspect , the present invention provides a use of the pathogen diagno s i s system, that is, a diagno s i s method of pathogen . I n particular , the present invention provides a diagno s i s method of pathogen for identifying pathogen (s) i nvo 1ved in developing infection wherein there are a plural of pathogens which may be involved.

Specifically, as shown in FIG. 10 , the method comprises : a body fluid collecting step (S20) for collecting part of body fluid being provided from a test body and containing DNA ; a DNA extraction step ( S 30 ) for extracting DNA from the collected body fluid to obtain DNA extract ; a DNA ampl i fication reaction step ( S 40 ) for conducting DNA ampl i fication reaction in micro f low channel s being configured in a substrate containing at least the s ame number of micro f low channels a s the number of pathogens or of groups of pathogens to be detected, in the mi cr o f low channels the DNA extract obtained in the step ( S 30 ) , and the PGR reagent including thermostable DNA polymerase , buf f er , deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the ampl i f i ed DNA being loaded, and primers be i ng introduced prior to the DNA ampl i fication reaction in each of the micro f 1 ow channel s in a manner that one kind of primer s is ne er introduced in two or more micro flow channel s , corresponding to each of a plural pathogens and being for amplifying each of target DNAs having sequence distinguishing each of the pathogens ; and a detecting step ( S 50 ) for detecting labeling agent which reacts with the produced DNA by the DNA amplification reaction, wherein the pathogen corresponding to a primer being loaded in the micro f 1 ow channel in which the labeling agent is detected in the detecting step ( S 50 ) is identified a s a pathogen involved in developing infection (S60) .

[ 0056 ]

I n FIG. 10 , in the step ( S 10 ) , a substrate is prepared in a manner that primers are introduced in mi cro flow channel s . I n particular, a s mentioned above , the primer reagent s are introduced from prescribed positions of the substrate corresponding to each of the micro flow channel s , and then the substrate is sub ected to drying . At this time, it must be noted that one primer should not be introduced in two of more micro flow channels; that is, each of the micro flow channels contains the different primer ( s ) from one another .

[0057]

I n the step ( S 20 ) , a body fluid (for example , muco sal fluid) of a patient as a test body is sampled by a body fluid collecting means .

[ 0058 ]

I n the step ( S 30 ) , the body fluid sampled in the step ( S 20 ) is introduced and mixed in a DNA extraction container with a DNA extraction reagent , to thereby conduct the DNA extraction reaction and obtain a DNA extract . To the DNA extraction container may be introduced together a PGR reagent together prior to the introduction of the body fluid a s ment ioned above . Further , in the DNA extraction reaction, the DNA extraction container into which the DNA extraction reagent and the body fluid are introduced may be heated or cooled ( or regulated ) , so that the reaction may be initiated, promoted or terminated.

[ 0059 ]

In the step (S40), the DNA extract obtained in the step ( S 30 ) is collected by use of an extract collecting means , and then introduced from a feed for the extract into the micro flow channel s conf igured on the substrate being prepared in the step ( S 10 ) . Thereafter , the heating and cooling condition for the substrate is controlled in a manner that the temperature profile a s shown in FIG. 7 is repeated for prescribed time s , so that the micro f low channels are sub j ected to the condition for amplifying DNA .

Further , at the time , one of the micro f low channels may be used a s a positive control . Furthermore , one of the rest of the mi cro f low channel s may be used a s a negati e control .

I n controlling the heating and cooling conditions , a steam thermal cycler (heating and cooling apparatus ) may be used, so that heating by steam and cooling by dry air for the substrate are conducted .

[0060]

I n the step ( S 50 ) , the detection is conducted for recognizing if the ampl i fication reaction is occurred by the heating and cooling operation on the substrate being conducted in the step (S40) ; that is, the labeling agent which is reacted with the ampl i f i ed DNA is detected . Detecting the labeling agent ( s ) indicates that the DNA ampl i fication reaction takes oo place in the detected micro flow channel ( s ) . Further , in the detection, a spectrometer may optionally be used.

[0061]

I n the step ( S 60 ) , the micro flow channel ( s ) in which the 1 abe 1 i ng agent is detected in the step ( S 50 ) is ident i f ied , so that the di agno s i s is conducted by identifying the pathogen ( s ) , corresponding to the primer ( s ) which is introduced in the detected micro flow channel, as pathogen(s) involved in developing infection in the test body who provides the body fluid . Then , the diagno s i s is finished. I f no labeling agent is detected from any micro flow channel, the diagnosis is conducted to recognize that no pathogen, corresponding to any of primer being introduced in the micro flow channel , involves in developing infection in the test body .

[ 0062 ]

From further aspect , the present invention provides a DNA amplifying kit which is suitably used for the pathogen diagno s i s system, in particular , a DNA amplifying kit for amplifying a specific target DNA ( s ) in parallel which exists in a sample containing DNA .

Specifically, the kit comprises : a s ampl e collecting means for collecting part of a s ampl e containing DNA, such as a body fluid from a patient with infection ; a DNA extraction reagent for extracting DNA from the collected sample to obtain DNA extract; a PGR reagent for amplification reaction of DNA on the DNA extract, comprising thermostable DNA polymerase, buffer, deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with the amplified DNA; a plural types of primer reagents each compr i sing one or more kinds of prime r s for amp lification of target DNAs, each primer corresponding to each of the target DNAs; a DNA extraction container for providing a field for DNA extraction reaction by use of the DNA extraction reagent; an extract collecting means for collecting part of DNA extract obtained in the DNA extraction container; and a substrate containing at least the same number of micro flow channels as the number of the target DNAs or of groups thereof , in the mi cro flow channels the DNA extract collected by the extract collecting means, the PGR reagent and the primer reagent being loaded, and a field for DNA amplification reaction being provided, wherein primer (s), that is, a primer or a group of primers is allowed to be introduced in each of the micro flow channel s , and wherein one primer is not introduced in two or more micro flow channel s . Further , the kit may contain an instruction for guiding an operator in such a way of operating the kit in a protocol in the order of the steps (S10) to (S40), and steps ( S 50 ) and ( S 60 ) , as shown in FIG. 10 , if needed .

Moreover , when the kit is used in a di agno s i s for infection, in whi ch the pathogen ( s ) to be sub ected to the detection, and DNA ( s ) to be amplified is predetermined, the kit may be provided in such a manner that the primer reagent ( s ) is introduced in advance in mi cr o flow channe 1 ( s ) of the substrate .

Accordingly, the operator only operates the kit in starting with the step ( S 20 ) of FIG. 10 , so that the ampl i fication of DNA and diagnosis for identifying the pathogen ( s ) of infection may be conducted . I n such a case , the kit may contain an instruction for guiding an operator in such a way of operating the kit .

EXAMPLES

[ 0063 ]

The present invention is explained in greater detail using the examples below. The present invention is not limited to these example s .

[0064]

( Example 1 )

The following bacteria a s known for typi cal pathogenic bacteria of pneumonia are focused. DNA fragment s a s shown in the following t able corresponding to each of the bacteria (both of "FORWARD" direction ampl i fication and "REVERSE" direction ampl i fication of the DNAs specific to each of the bacteria ) were employed a s suitable primers for DNA ampl i fication by the PGR method of those bacteria . [0065]

Table 1

MSSA : Methicillin-Sensitive Staphylococcus Aureus

MRSA : Methicillin-Resistant Staphylococcus Aureus [ 0066 ]

A substrate of borosilicate glass configured a s shown in FIG . 1 ( CAs-CHIP; manufactured by METABOSCREEN CO . , LTD . ) was sterilized. Thereafter, to each of seven micro f 1 ow channels out of eight were separately introduced the pr imer s ( both of FORWARD and REVERSE ) corresponding to each of the bacteria as shown in Table 1 , respectively . To the rest of one micro f 1 ow channel was introduced a primer pair who s e target gene is human bet a-gl obi n (bglo-F&R) for exhibiting the positive control . Each reagent was set in the eight of micro flow channels, and the substrate was sub ected to drying, to thereby obtain a primer-containing substrate in whi ch the prime r s were introduced into the micro f 1 ow channels .

The dimension of the substrate was 10 mm in diameter and 0.3 mm in thickness; and the dimension of each of the mi cro f low channel s was 5 mm in length, 0.1 mm in width and 0.1 mm in depth .

[0067] ( Example 2 )

From the test body who developed pneumonia, one platinum loop of purulent sputum was sampled. The sampled purulent sputum was mixed in a tube together with CellEase ( tm ) I I ( available from Biocosm Inc . ) used as a DNA extraction reagent . I n the tube , prior to introducing the purulent sputum, a PGR reagent containing thermostable DNA polymerase and buf fer for reaction ( TaKaRa Ex Taq (tm), available from TAKARA BIO INC . ) and a spectroscopically detectable 1 abe 1 i ng agent (SYBR ( tm ) Green I , avail able from TAKARA BIO INC . ) were already mixed .

[0068]

( Example 3 )

The DNA extract thus obtained in Example 2 was dropped onto a feed for the extract of the primer-containing substrate prepared in Example 1 , to thereby be introduced into each of the mi cro f low channel s . Subsequently, the substrate was sub ected to the heating and cooling treatment by a steam heating and cooling apparatus as shown in FIGs . 8 and 9 under the condition for repeating 35 time s of a thermal cycle wherein in accordance with the temperature profile a s shown in FIG. 7 the annealing temperature (T_L) was set to 60 degrees C ; the ampl i fication temperature (T_M) to 72 degrees C ; and the denaturing temperature ( T_H ) to 95 degr ee s C ; each period of time was set to the following table .

[0069] Table 2

[0070]

( Example 4 )

The substrate which was subjected to the condition for the DNA amplification reaction in Exampl e 3 was irradiated with an exciting light at the center of the wavelength of 450 nm ( cut from 500 nm or longer ) and irradiation area of 10 mm s quare or larger , and then with a detecting light at the center of the wavelength of 520 nm ( cut from 500 nm or shorter ) , and then took an image of the surface of the substrate , to thereby detect if the DNA ampl i fication reaction took place .

[0071]

( Example 5 )

Since the DNA ampl i fication reaction was detected in Exampl e 4 at the micro f 1 ow channel in which the primer corresponding to Staphylococcus aureus (MRSA) had been introduced, Staphylococcus aureus (MRSA) was identified as a pathogen involved in developing pneumonia. EXPLANATIONS OF LETTERS OR NUMERALS

[0072]

11, lib : Feed for DNA Extract

11a: Primer Feed

20: Heating and Cooling Apparatus

21a to 21h: Micro Flow Channel

22a to 22s: Micro Flow Channel

23a to 23s: Micro Flow Channel

31: DNA Extract

32 : Ai r Layer

33 : Mineral Oil

40, 41: Substrate

50 : Substrate Holder

51a: Guide

51b, 51c: Opening

52 : Sealing Material Feed

53 : Open Space for Sealing Material

60: Motor

60a: Axi s

60b: Supporting Board

61 : Steam Noz z le 62, 63: Air Nozzle

70: Control Unit

71: Steam Generator

72, 73 : Blower

SEQUENCE LISTING

<110> Daiken Medical Co., LTD.

<120> DIAGNOSIS METHOD OF PATHOGEN

<130> TEK/FP6882153

<160> 14

< 170 > Patentln version 3.5

<210> 1

<211> 21

<212> DNA

< 213 > Artificial Sequence

<220>

<223> Primer for PGR

<400> 1

caaccgtaca gaatgaagcg g

21

<210> 2

<211> 22

<212> DNA

< 213 > Artificial Sequence

<220>

<223> Primer for PGR

<400> 2

ttattcgtgc aatactcgtg eg

22

<210> 3 < 211 > 21

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PGR

<400> 3

ttgacatcct aagaagagct c 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PGR <400> 4

tctcctttga gttcccgacc g 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PGR

<400> 5

gcgattgatg gtgatacggt t 21

<210> 6

<211> 25

<212> D A

<213 > Artificial Sequence <220>

<223> Primer for PGR

<400> 6

agccaagcct tgacgaacta aagcc 25

<210> 7

<211> 21

<212> DNA

<213 > Artificial Sequence <220>

<223> Primer for PCR

<400> 7

tatccaccct caaacaggtg a 21

<210> 8

<211> 21

<212> DNA

<213 > Artificial Sequence <220>

<223> Primer for PCR <400> 8

ctggaacttg ttgagcagag g 21

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PGR <400> 9

atacagtggt cagtgatcgc c 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PGR

<400> 10

agaagtacca ttaccaccgc c 21

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PCR

<400> 11

cacttcatcg gctacatcta eg 22

<210> 12

<211> 22

<212> DNA

< 213 > Artificial Sequence <220>

<223> Primer for PCR

<400> 12

agatattege tctggtaggt gg 22

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence <220>

<223> Primer for PCR

<400> 13

caaccgatgc cacatcatta 20

<210> 14

<211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Primer for PGR <400> 14

atagcgtctt gcatgccttt

20

Claims

What is claimed:

1. A diagnosis method of pathogen for identifying pathogen(s) involving in developing infection wherein there are a plural of pathogens which may be involved, comprising :

A body fluid collecting step for collecting part of body fluid being provided from a test body and containing DNA;

A DNA extraction step for extracting DNA from the collected body fluid to obtain DNA extract;

A DNA amplification reaction step for conducting DNA ampl i fication reaction in micro flow channel s be i ng conf igur ed in a substrate containing at least the same number of mi cro f low channel s as the number of pathogens or of groups of pathogens to be detected, in the micro f low channels the DNA extract obtained in said DNA extraction step, and a PGR reagent including thermostable DNA polymerase , buffer ,

deoxynucleoside triphosphates, and labeling agent being spectroscopically detectable by reacting with ampl i f i ed DNA being loaded, and primers being introduced prior to DNA ampl i fication reaction in each of said mi cro f low channel s in a manner that one kind of primers is never introduced in two or more micro f 1 ow channel s , corresponding to each of a plural pathogens and being for amplifying each of target DNAs having sequence distinguishing each of said pathogens ; and

A detecting step for detecting labeling agent which reacts with the produced DNA by the DNA amplification reaction, Wherein the pathogen corresponding to a primer being loaded in the micro flow channel in which the labeling agent is detected in said detecting step is identified a s a pathogen i nvo 1 i ng in de eloping infection .

2. The di agno s i s method a s claimed in Claim 1 , wherein said substrate further comprises a micro f low channel for a positive control .

3. The di agno s i s method a s claimed in Claim 2 , wherein said substrate further c ompr i s e s a mi cr o flow channel for a negative control .

4. The di agno s i s method a s claimed in any of Claims 1 to 3 , wherein a temperature control for the DNA amplification reaction is conducted by heating by s team and cooling by dry air said substrate in said DNA ampl i fication reaction step .

5. The di agno s i s method a s claimed in any of Claims 1 to 4 , wherein said PGR reagent is introduced together with said DNA extraction reagent in extraction reaction conducted in said DNA extraction step .