在BACTEC 12B培养物中，使用聚合酶链反应替代rho-硝基-α-乙酰氨基-β-羟基苯丙酮试验用于早期检测结核分枝杆菌的替代方法。

Alternative use of polymerase chain reaction instead of rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone test for the early detection of Mycobacterium tuberculosis in BACTEC 12B cultures.

作者信息

Ryang D W, Ryang D H, Shin M G, Shin J H, Kee S J, Suh S P

机构信息

Department of Clinical Pathology, Chonnam University Medical School, Dong-ku, Kwangju, Republic of Korea.

出版信息

APMIS. 1996 Jun;104(6):444-50.

PMID:8774674

Abstract

Compared with conventional culture media, the TB BACTEC system has demonstrated improved isolation rates as well as an earlier detection time for mycobacterial species. However, the identification of M. tuberculosis by the rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test in the TB BACTEC 460 system may require 6 days for interpretable results. We evaluated the usefulness of a polymerase chain reaction (PCR) assay for earlier identification of M. tuberculosis in positive BACTEC 12B cultures. A total of 262 TB BACTEC culture specimens with GIs > or = 10 were assayed by PCR, and the results were compared with those of the NAP test. The aliquot from BACTEC 12B vials was boiled for 10 min, and 2 microliters of the boiled suspension was used for the PCR assay. One set of primers based on the IS 6110 sequence of M. tuberculosis was used to amplify a 457 bp fragment of DNA. Of the 173 TB BACTEC culture specimens which were identified as M. tuberculosis by the NAP test. 171 were PCR positive. Of the 21 TB BACTEC cultures identified as MOTT by the NAP test. 19 were PCR negative, but 2 were PCR positive: these two cultures were shown to grow both M. tuberculosis and MOTT in BACTEC 12B vials. Of the remaining 68 cultures which were contaminated with AFB-negative bacteria, the PCR identified M. tuberculosis in 13, in agreement with the NAP results in the reprocessed specimens. Overall, the PCR results in the 262 BACTEC culture specimens with GIs > or = 10 were sensitive in 99.5% (186/187) and specific in 100% (68/68). The mean time for the identification of M. tuberculosis in TB BACTEC cultures with GIs > or = 10 was 7 h by the PCR compared to 5.9 days by the NAP test. These results suggest that the PCR could be used as an alternative to the NAP test for the rapid identification of M. tuberculosis in BACTEC 12B cultures, particularly in those which contained both M. tuberculosis and MOTT or M. tuberculosis and AFB-negative bacteria.

摘要

与传统培养基相比，结核分枝杆菌BACTEC系统已显示出提高的分离率以及更早的分枝杆菌属检测时间。然而，在结核分枝杆菌BACTEC 460系统中通过rho-硝基-α-乙酰氨基-β-羟基苯丙酮（NAP）试验鉴定结核分枝杆菌可能需要6天才能获得可解释的结果。我们评估了聚合酶链反应（PCR）检测法在结核分枝杆菌BACTEC 12B阳性培养物中早期鉴定结核分枝杆菌的实用性。对总共262份生长指数（GI）≥10的结核分枝杆菌BACTEC培养标本进行了PCR检测，并将结果与NAP试验结果进行比较。从BACTEC 12B小瓶中取出的等分试样煮沸10分钟，取2微升煮沸后的悬液用于PCR检测。使用一组基于结核分枝杆菌IS 6110序列的引物扩增457bp的DNA片段。在通过NAP试验鉴定为结核分枝杆菌的173份结核分枝杆菌BACTEC培养标本中，171份PCR检测呈阳性。在通过NAP试验鉴定为非结核分枝杆菌（MOTT）的21份结核分枝杆菌BACTEC培养物中，19份PCR检测呈阴性，但2份PCR检测呈阳性：这两份培养物在BACTEC 12B小瓶中同时培养出结核分枝杆菌和MOTT。在其余68份被抗酸杆菌阴性细菌污染的培养物中，PCR在13份中鉴定出结核分枝杆菌，与重新处理标本后的NAP结果一致。总体而言，在262份GI≥10的BACTEC培养标本中，PCR结果的敏感性为99.5%（186/187），特异性为100%（68/68）。通过PCR鉴定GI≥10的结核分枝杆菌BACTEC培养物中结核分枝杆菌的平均时间为7小时，而通过NAP试验则为5.9天。这些结果表明，PCR可作为NAP试验的替代方法，用于快速鉴定结核分枝杆菌BACTEC 12B培养物中的结核分枝杆菌，特别是在那些同时含有结核分枝杆菌和MOTT或结核分枝杆菌和抗酸杆菌阴性细菌的培养物中。