Zwadyk P, Down J A, Myers N, Dey M S
Veterans Affairs Medical Center, Durham, North Carolina 27705.
J Clin Microbiol. 1994 Sep;32(9):2140-6. doi: 10.1128/jcm.32.9.2140-2146.1994.
Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens.
在用DNA扩增方法检测分枝杆菌标本之前，必须满足两个标准：（i）样本必须进行无害化处理，（ii）必须裂解微生物以释放DNA。以往报道分枝杆菌DNA扩增的文献主要集中在裂解和扩增程序上，未涉及样本安全性问题。我们已经表明，在低于100摄氏度的温度下加热样本可能无法始终如一地杀死分枝杆菌；然而，在沸水浴或强制通风烘箱中于100摄氏度加热至少5分钟可杀死分枝杆菌，包括耐热分枝杆菌。此外，在100摄氏度加热30分钟可始终如一地裂解分枝杆菌，产生适合通过PCR和链置换扩增进行扩增的短DNA片段。该程序适用于经N-乙酰半胱氨酸-NaOH方法消化的临床样本以及磷酸盐缓冲液中的微生物悬液。本文还证明了对临床标本使用链置换扩增的可行性。
