Kit for detection of microorganism

Abstract

A kit for use in a method for detecting live cells, injured cells and dead cells of a microorganism in a test sample by a nucleic acid amplification method is disclosed. The kit includes a cross-linker capable of cross-linking DNA by irradiation with light having a wavelength of 350 nm to 700 nm, medium, and a primer (s) for amplifying a DNA target region in the microorganism by a nucleic acid amplification method. The nucleic acid amplification method may be a PCR, LAMP, SDA, LCR or DNA microarray method. A cross-linker may be included in the kit such as ethidium monoazide, ethidium diazide, psoralen, 4,5′,8-trimethyl psoralen, or 8-methoxy psoralen.

1 . A kit for use in a method for detecting live cells, injured cells and dead cells of a microorganism in a test sample by a nucleic acid amplification method, which comprises the following components:
 a cross-linker capable of cross-linking DNA by irradiation with light having a wavelength of 350 nm to 700 nm;   a medium; and   a primer(s) for amplifying a target region of a DNA of a microorganism, which is a detection target, by the nucleic acid amplification method.   
     
     
         2 . The kit according to  claim 1 , wherein the method comprises the following steps:
 a) adding the cross-linker capable of cross-linking DNA by irradiation with light having a wavelength of 350 nm to 700 nm to the test sample;   b) irradiating the test sample to which the cross-linker is added with light having a wavelength of 350 nm to 700 nm;   c) removing the cross-linker contained in the test sample irradiated with light;   d) adding a medium to the test sample from which the cross-linker is removed and incubating the test sample;   e) adding again the cross-linker capable of cross-linking a DNA by irradiation with light having a wavelength of 350 nm to 700 nm to the incubated test sample;   f) irradiating the test sample to which the cross-linker is added with light having a wave length of 350 nm to 700 nm;   g) extracting a DNA from the test sample and amplifying a target region of the extracted DNA by a nucleic acid amplification method; and   h) analyzing the amplified product.   
     
     
         3 . The kit according to  claim 1 , wherein the nucleic acid amplification method is a PCR, LAMP, SDA, LCR or DNA microarray method. 
     
     
         4 . The kit according to  claim 1 , wherein the cross-linker is selected from the group consisting of ethidium monoazide, ethidium diazide, psoralen, 4,5′,8-trimethyl psoralen, and 8-methoxy psoralen. 
     
     
         5 . The kit according to  claim 2 , wherein the nucleic acid amplification method is a PCR, LAMP, SDA, LCR or DNA microarray method. 
     
     
         6 . The kit according to  claim 2 , wherein the cross-linker is selected from the group consisting of ethidium monoazide, ethidium diazide, psoralen, 4,5′,8-trimethyl psoralen, and 8-methoxy psoralen. 
     
     
         7 . The kit according to  claim 3 , wherein the cross-linker is selected from the group consisting of ethidium monoazide, ethidium diazide, psoralen, 4,5′,8-trimethyl psoralen, and 8-methoxy psoralen. 
     
     
         8 . The kit according to  claim 5 , wherein the cross-linker is selected from the group consisting of ethidium monoazide, ethidium diazide, psoralen, 4,5′,8-trimethyl psoralen, and 8-methoxy psoralen.