Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India.
Jpn J Infect Dis. 2010 Jan;63(1):55-7.
We attempted to apply the PCR-restriction fragment length polymorphism (PCR-RFLP) technique for the early detection and identification of Mycobacterium tuberculosis directly from clinical samples. PCR-RFLP of hsp65 was applied to the DNA extracted directly from sputum samples (n = 226) collected from 226 patients. We could detect and identify M. tuberculosis in 84.5% of the acid-fast bacillus (AFB) smear-positive samples (n = 149) and 11% of the AFB smear-negative samples (n = 18) obtained from patients with clinical and radiological evidence of tuberculosis. Sputum samples (n = 59) obtained from patients suffering from respiratory diseases other than tuberculosis were included as negative controls. To test the sensitivity of the assay, we spiked a smear-negative sample with serial dilutions of H37Rv. The protocol could detect down to 10 organisms/microl. PCR-RFLP was found to be a simple and reproducible method for early detection of M. tuberculosis from sputum samples. The present assay could be used to augment conventional methods of diagnosis of mycobacterial diseases and thus help clinicians to differentiate between M. tuberculosis complex and non-tuberculous mycobacteria directly in clinical samples. The assay could help clinicians to select appropriate chemotherapeutic agents early, which could considerably reduce the morbidity due to mycobacterial diseases.
我们试图应用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术直接从临床样本中早期检测和鉴定结核分枝杆菌。将 hsp65 的 PCR-RFLP 应用于直接从 226 名患者的痰样本中提取的 DNA（n = 226）。我们可以在 84.5%的抗酸杆菌（AFB）涂片阳性样本（n = 149）和 11%的 AFB 涂片阴性样本（n = 18）中检测和鉴定结核分枝杆菌，这些样本来自具有临床和影像学证据的结核病患者。将患有除结核病以外的呼吸道疾病的患者的痰样本（n = 59）作为阴性对照。为了测试该检测方法的灵敏度，我们用 H37Rv 的连续稀释液对 AFB 涂片阴性样本进行了加标。该方案可检测到低至 10 个/微升的生物量。PCR-RFLP 是一种从痰样本中早期检测结核分枝杆菌的简单且可重复的方法。该检测方法可用于增强分枝杆菌病的常规诊断方法，从而帮助临床医生直接在临床样本中区分结核分枝杆菌复合群和非结核分枝杆菌。该检测方法可以帮助临床医生早期选择合适的化疗药物，这可以大大降低因分枝杆菌病导致的发病率。
