痰简易处理法结合巢式PCR在检测结核杆菌中的应用

SIMPLE METHOD FOR SPUTUM SAMPLES COMBINED WITH NESTED POLYMERASE CHAIN REACTION IN DETECTION OF MYCOBACTERIUM TUBERCULOSIS

作者对106例结核性痰标本采用简易处理法（TET煮沸法）后，再用聚合酶链反应（PCR）检测，阳性率（44．3％）与酚／氯仿抽提法（46．2％）相近（X2＝0．076，P＞0．05），巢式PCR（nested－PCR）阳性率（72．6％）则显著高于PCR（X2＝17．485，P＜0．01），这种差异在涂片和培养均阴性的标本中尤为明显。巢式PCR扩增人结核菌DNA，其敏感性（50fg）为PCR的100倍，但不影响其特异性。21例非结核性痰标本经PCR和巢式PCR检测均为阴性。因而我们认为，TET煮沸法作为痰PCR前处理简易有效，对含菌量较小的标本有必要结合巢式PCR，以提高结核杆菌阳性诊断率。

A simple method (TET-boiling method )was used to treat 106 sputum samples compared with the typical phenolchloroform extraction of DNA. They yielded similar positive rates (44.3%vs 46.2%) of polymerase chain reaction (PCR), while nested PCR had a much higher one (72.6%) with the greatest difference from those with smear-negative and culture-negative results.Using purified DNA of Mycobacterium tuberculosis,we also determined the sensitivity (50fg) of nested PCR,which was 100 times as sensitive as PCR. But it didn't affect the specificity of PCR. PCR and nested PCR were all negative in 21 nontuberculous sputum samples. We conclude that the TET-boiling method is a simple and effective DNA extraction procedure,and it should be combined with nested PCR in detecting samples with very small amount of bacteria.