建立增菌PCR方法对痰涂片阴性肺结核的早期诊断

Accelerated diagnosis of smear-negative pulmonary tuberculosis with bacteria proliferation accompanied PCR(BPAP)

目的 建立增菌PCR方法 ,以达到对涂片阴性痰标本中结核菌早期检定和确认活菌的目的。方法 采用 7H12半流休培养和PCR扩增相结合的增菌PCR方法 ,对 4 6份初治肺结核患者痰标本进行检测 ,并对杂交结果进行凝胶呈像灰度分析。结果 应用增菌PCR检测涂片阴性痰标本 ,其中 86 .2 %的标本在增菌 7d内得到阳性结果 ,于 4周时得到最大阳性率 ,较培养法的阳性检出时间明显缩短 (P <0 .0 1) ,较普通标本PCR阳性率明显提高 (P <0 .0 5 ) ;有 6 3.0 %的标本增菌 7d时的杂交结果凝胶灰度值与 0d时相比 >1;并有 6 2 .1%的增菌PCR 7d内得到阳性的标本得到同一样本7H12半流培养阳性结果的支持和活菌判定。结论 增菌PCR不是单纯的PCR ,增菌 7d后PCR阳性率明显增加 ,且有杂交结果凝胶灰度比值的增加和随后的培养结果对其进行支持和活菌判断 ,有助于临床诊断的确切性。

Objective To develop a new method for detecting smear-negative sputum from patients with pulmonary tuberculosis,for the objectives of rapidly detection and determine viable bacillus.Methods By using a new method,called bacteria proliferation accompanied PCR(BPAP),which combine culture with Middlebrook 7H12 semi-liquid media and PCR,46 specimens were detected.Their Southern-blot results were analyzed by Gel-Pro Analyzer software.Results (1)For smear-negative specimens,to the 7th day,the sensitivity of BPAP was 86.2%;To 4 weeks,BPAP reached its highest sensitivity.It needed a shorter time to the detection of M.tuberculosis for BPAB than culture(P<0.01);And it had a higher sensitivity for BPAP than common PCR(P<0.05).(2)Out of 46 specimens ,there were 29 specimens(63.0%)that Southern-blot IOD ratio between the 7th day and the Oth day>1 zero;And for those specimens got their positive BPAP results within 7 days,62.1% were supported by the positive 7H12 semi-liquid cultuer results.Conclusion BPAP is not common PCR.Through 7 days' bacteria proliferation,there is a significant increase in the sensitivity.And for the support and determination of viable organisms by culture and Southern-blot IOD ratio.BPAP helps to get a more accurate clinical diagnosis.