Method for detecting microorganisms

Abstract

The present invention relates to a method for simultaneously detecting a plurality of pathogens from biologically-derived samples, and a kit for carrying out the method. Specifically, the present invention relates to a method for simultaneously detecting a plurality of pathogens that cause infectious uveitis, one of eye infections from samples such as anterior chamber fluid or vitreous by polymerase chain reaction (PCR), and a kit for carrying out the method.

1 . A method for detecting microorganisms in a sample, comprising
 (1) a step of mixing the sample with a PCR buffer containing a surfactant;   (2) a step of adding a part of the mixed solution obtained in the step (1) to a tube containing a solid composition for PCR reaction, which is a tube strip formed by connecting a plurality of tubes, and each tube contains DNA polymerase and one or more types of PCR primer pairs; and   (3) a step of detecting the PCR product generated in the tube.   
     
     
         2 . The method according to  claim 1 , wherein the sample is anterior chamber fluid or vitreous. 
     
     
         3 . The method according to  claim 1 , wherein the amount of the sample is 12-20 μL. 
     
     
         4 . The method according to  claim 1 , wherein the microorganism is selected from the group consisting of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), cytomegalovirus (CMV), human adult T-cell leukemia virus (HTLV-1),  Treponema pallidum  and  toxoplasma.    
     
     
         5 . The method according to  claim 1 , wherein in the step (1), the surfactant is a nonionic surfactant. 
     
     
         6 . The method according to  claim 1 , wherein in the step (1), the PCR buffer is Tris buffer containing KCl, MgCl 2  and dNTP mix (a mixture of dATP, dGTP, dCTP and dTTP). 
     
     
         7 . The method according to  claim 1 , wherein in the step (1), the PCR buffer binds to substances which are biologically-derived negatively charged substance that adsorbs to DNA polymerase and biologically-derived a positively-charged substance that adsorbs to DNA, and inhibit PCR and contains a substance that neutralizes the PCR inhibitory effect of the negatively charged substance and the positively charged substance. 
     
     
         8 . The method according to  claim 1 , wherein in the step (2), the tube strip is 2-12 tube strips. 
     
     
         9 . The method according to  claim 1 , wherein in the step (2), the PCR primer pairs are those for detecting herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), Cytomegalovirus (CMV), human adult T-cell leukemia virus (HTLV-1),  Treponema pallidum  or  toxoplasma.    
     
     
         10 . The method according to  claim 1 , wherein in the step (2), the two types of PCR primer pairs contained in each tube are the following combinations.
 (i) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene detection primer pair and TATA-binding protein (TBP) gene detection primer pair   (ii) HSV-1 detection primer pair and VZV detection primer pair   (iii) HSV-2 detection primer pair and HHV-6 detection primer pair   (iv) EBV detection primer pair and CMV detection primer pair   (v) HTLV-1 detection primer pair and  Treponema pallidum  detection primer pair.   
     
     
         11 . The method according to  claim 1 , wherein in the step (2), the solid composition for PCR reaction contains an oligonucleotide probe labeled with one or more types of fluorescent dyes for fluorescent detection of PCR amplification products. 
     
     
         12 . The method according to  claim 11 , wherein the fluorescent dyes are selected from the group consisting of FAM (6-carboxyfluorescein), ROX (6-carboxy-X-rhodamine), Cy5 (Cyanine dye) and HEX (4,7,2′,4′,5′,7′-hexachlorofluor-6-carboxyfluorescein). 
     
     
         13 . The method according to  claim 1 , wherein in the step (2), the solid composition for PCR reaction is prepared by lyophilization. 
     
     
         14 . The method according to  claim 1 , wherein in the step (3), the PCR products are detected by real-time determination. 
     
     
         15 . A kit for detecting microorganisms in a sample, equipped with tube  1  which contains a PCR buffer containing a surfactant for mixing the sample, and tube  2  which contains solid composition for PCR reaction which is a tube strip formed by connecting a plurality of tubes, and each tube contains DNA polymerase and one or more types of PCR primer pairs for adding a part of the mixed solution contained in tube  1 . 
     
     
         16 . The kit according to  claim 15 , wherein the surfactant is a nonionic surfactant. 
     
     
         17 . The kit according to  claim 15 , wherein the PCR buffer is Tris buffer containing KCl, MgCl 2  and dNTP mix (mixture consisting of dATP, dGTP, dCTP and dTTP). 
     
     
         18 . The kit according to  claim 15 , wherein the PCR buffer, wherein the PCR buffer binds to substances which are biologically-derived negatively charged substance that adsorbs to DNA polymerase and biologically-derived a positively-charged substance that adsorbs to DNA, and inhibit PCR and contains a substance that neutralizes the PCR inhibitory effect of the negatively charged substance and the positively charged substance. 
     
     
         19 . The kit according to  claim 15 , wherein the strip tube is 2-12 tube strip. 
     
     
         20 . The kit according to  claim 15 , wherein the PCR primer pairs are those for detecting herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), Cytomegalovirus (CMV), human adult T-cell leukemia virus (HTLV-1),  Treponema pallidum  or  toxoplasma.    
     
     
         21 . The kit according to  claim 15 , wherein the two types of PCR primer pairs contained in each tube are the following combinations:
 (i) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene detection primer pair and TATA-binding protein (TBP) gene detection primer pair   (ii) HSV-1 detection primer pair and VZV detection primer pair   (iii) HSV-2 detection primer pair and HHV-6 detection primer pair   (iv) EBV detection primer pair and CMV detection primer pair   (v) HTLV-1 detection primer pair and  Treponema pallidum  detection primer pair.   
     
     
         22 . The kit according to  claim 15 , wherein the solid composition for PCR reaction contains an oligonucleotide probe labeled with one or more types of fluorescent dyes for fluorescent detection of PCR amplification products. 
     
     
         23 . The kit according to  claim 22 , wherein the fluorescent dyes are selected from the group consisting of FAM (6-carboxyfluorescein), ROX (6-carboxy-X-rhodamine), Cy5 (Cyanine dye) and HEX (4,7,2′,4′,5′,7′-hexachlorofluor-6-carboxyfluorescein). 
     
     
         24 . The kit according to  claim 15 , wherein the solid composition for PCR reaction is prepared by lyophilization. 
     
     
         25 . A method for testing a gene, comprising
 (1) a step of mixing a sample and a PCR buffer containing a surfactant;   (2) a step of adding a part of the mixed solution obtained in the above step (1) to a solid composition for PCR reaction which contains DNA polymerase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) detection primer pair and/or TATA-binding protein (TBP) detection primer pair;   (3) a step of adding a part of the mixed solution obtained in the above step (1) to a solid composition for PCR reaction which contains DNA polymerase and one or more types of PCR primer pairs; and   (4) a step of detecting the PCR products generated as results of the steps (2) and (3).