Method of effecting lysis of acid-fast bacteria and method of performing gene amplification or detection therewith

Abstract

A method of effecting lysis of acid-fast bacteria, comprising heating acid-fast bacteria in a liquid containing a non-ionic surfactant at a temperature of below the boiling point of the liquid. This method enables accomplishing secure lysis of acid-fast bacteria in a simple manner within a short period of time without the use of special apparatus and agent and enables extracting genes. The heating is preferably conducted at 96° C. for 10 min. As the nonionic surfactant, use can be made of a d-sorbitol fatty acid ester, a polyoxyethylene glycol sorbitan alkyl ester, a polyoxyethylene glycol p-t-octylphenyl ether or the like. The pH value of the liquid is preferably 8, and the liquid preferably contains EDTA. It is also preferred that before the heating, the acid-fast bacteria be treated with lipase.

1 . A method of lysing an acid-fast bacterium to extract a gene from the acid-fast bacterium, comprising: 
 heating the acid-fast bacterium in a liquid containing a non-ionic detergent at a temperature below a boiling point of the liquid.    
     
     
         2 . The method according to  claim 1 , wherein the heating temperature is not less than 70° C. and less than 100° C.  
     
     
         3 . The method according to  claim 1 , wherein the heating is performed for 1 to 30 minutes.  
     
     
         4 . The method according to  claim 1 , wherein the heating is performed at 96° C. for 10 minutes.  
     
     
         5 . The method according to  claim 1 , wherein a pH of the liquid is in a range from 7.0 to 12.0.  
     
     
         6 . The method according to  claim 1 , wherein a concentration of the non-ionic detergent in the liquid is 0.01 to 10 wt %.  
     
     
         7 . The method according to  claim 1 , wherein the non-ionic detergent is at least one selected from the group consisting of D-sorbitol fatty acid esters, polyoxyethyleneglycol sorbitan alkyl esters, and polyoxyethyleneglycol p-t-octylphenyl ethers.  
     
     
         8 . The method according to  claim 1 , wherein the liquid further contains a metal chelating agent.  
     
     
         9 . The method according to  claim 8 , wherein a concentration of the metal chelating agent in the liquid is 0.1 to 100 mM.  
     
     
         10 . The method according to  claim 8 , wherein the metal chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(β-aminoethyl ether)□N,N,N′,N′-tetraacetic acid (EGTA), diaminocyclohexane tetraacetic acid, o-phenanthroline, and salicylic acid.  
     
     
         11 . The method according to  claim 1 , wherein the acid-fast bacterium to be lysed is at least one selected from the group consisting of  M. avium, M. intracellularae, M. gordonae, M. tuberculosis, M. kansasii, M. fortuitum, M. chelonae, M. bovis, M. scrofulaceum, M. paratuberculosis, M. phlei, M. marinum, M. simiae, M. szulgai, M. leprae, M. xenopi, M. ulcerans, M. lepraemurium, M. flavescens, M. terrae, M. nonchromogenicum, M. malmoense, M. asiaticum, M. vaccae, M. gastri, M. triviale, M. haemophilum, M. africanum, M. thermoresistable,  and  M. smegmatis.    
     
     
         12 . The method according to  claim 1 , wherein a biological sample containing the acid-fast bacterium is at least one selected from the group consisting of sputum, spinal fluid, feces, saliva, blood, tissues, and urine.  
     
     
         13 . A method of amplifying or detecting a gene of an acid-fast bacterium specifically, comprising: 
 lysing an acid-fast bacterium by the method according to  claim 1  to extract a gene of the acid-fast bacterium; and    amplifying or detecting the gene specifically using the extracted gene as a sample.    
     
     
         14 . A method of lysing an acid-fast bacterium to extract a gene from the acid-fast bacterium, comprising: 
 causing lipolysis by treating the acid-fast bacterium with lipase, and    heating the acid-fast bacterium in the presence of a non-ionic detergent.    
     
     
         15 . The method according to  claim 14 , wherein the heating also serves to deactivate the lipase.  
     
     
         16 . The method according to  claim 14 , wherein the lipolysis and the heating are performed in a buffer.  
     
     
         17 . The method according to  claim 14 , wherein the lipolysis and the heating are performed in a same container as a closed system.  
     
     
         18 . The method according to  claim 14 , wherein the heating is performed after the lipolysis.  
     
     
         19 . The method according to  claim 18 , wherein the lipolysis is caused at a pH of 4 to 8 and at a temperature of 37° C. to 60° C. for 5 to 30 minutes, and the heating is performed at a temperature of 37° C. to 100° C. for 5 to 30 minutes.  
     
     
         20 . The method according to  claim 14 , wherein the lipolysis and the heating are performed simultaneously.  
     
     
         21 . The method according to  claim 20 , wherein the lipolysis and the heating are performed at a pH of 4 to 8 and at a temperature of 37° C. to 60° C. for 5 to 30 minutes.  
     
     
         22 . The method according to  claim 16 , wherein a concentration of the lipase in the buffer is 10 to 10000 units/ml.  
     
     
         23 . The method according to  claim 14 , wherein the non-ionic detergent is at least one selected from the group consisting of D-sorbitol fatty acid esters, polyoxyethyleneglycol sorbitan alkyl esters, and polyoxyethyleneglycol p-t-octylphenyl ethers.  
     
     
         24 . The method according to  claim 16 , wherein a concentration of the non-ionic detergent in the buffer is 0.01 to 10 wt %.  
     
     
         25 . The method according to  claim 14 , wherein the heating is performed in the presence of a metal chelating agent in addition to the non-ionic detergent.  
     
     
         26 . The method according to  claim 25 , wherein the metal chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), and 1,2-cyclohexanediaminetetraacetic acid (CyDTA).  
     
     
         27 . The method according to  claim 25 , wherein a concentration of the metal chelating agent in the buffer is 0.1 to 2.0 mM.  
     
     
         28 . The method according to  claim 14 , wherein the acid-fast bacterium to be lysed is at least one selected from the group consisting of  M. avium, M. intracellularae, M. gordonae, M. tuberculosis, M. kansasii, M. fortuitum, M. chelonae, M. bovis, M. scrofulaceum, M. paratuberculosis, M. phlei, M. marinum, M. simiae, M. szulgai, M. leprae, M. xenopi, M. ulcerans, M. lepraemurium, M. flavescens, M. terrae, M. nonchromogenicum, M. malmoense, M. asiaticum, M. vaccae, M. gastri, M. triviale, M. haemophilum, M. africanum, M. thermoresistable,  and  M. smegmatis.    
     
     
         29 . The method according to  claim 14 , wherein a biological sample containing the acid-fast bacterium is at least one selected from the group consisting of sputum, spinal fluid, feces, saliva, blood, tissues, swab, liquid obtained by gastrolavage, and urine.  
     
     
         30 . A method of amplifying or detecting specifically a gene of an acid-fast bacterium, comprising: 
 lysing an acid-fast bacterium by the method according to  claim 14  to extract a gene of the acid-fast bacterium; and    amplifying or detecting the gene specifically using the extracted gene as a sample.