Department of Laboratory Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan City, Taiwan.
Ph.D. Program in Biomedical Engineering, Chang Gung University, Taoyuan City, Taiwan.
Sci Rep. 2019 Jan 14;9(1):113. doi: 10.1038/s41598-018-33804-1.
Tuberculosis (TB) causes a heavy health burden worldwide, especially in developing countries. The need for the rapid and accurate diagnosis of TB has not been satisfied, especially for extra-pulmonary specimens or specimens with acid fast stain (AFS)-negative condition. Development and validation of a novel, sensitive and specific assay for diagnosing TB is essential. We developed IS4 primer/probe based on insertion sequence 6110 (IS6110). A qPCR assay was designed for detecting a specific region in IS6110 by BLAST. The IS4 primer/probe concentration, qPCR efficiency and various of PCR additives were evaluated and optimized. Thirty-four species of commonly isolated microorganisms were used for evaluating the analytical specificity. Moreover, 130 clinical specimens were collected for evaluating the performance versus Cobas TaqMan MTB (CTM) assay kit and culture. The amplification efficiencies of IS4 were 99.61% and 102.61% without and with internal control DNA (Bacteriophage Lambda), respectively. Dimethyl sulfoxide outperformed glycerol or BSA for eliciting the most effective amplification and the lowest limit of detection. In evaluating the clinical performance, various specimen types were collected. IS4 demonstrated a high degree of agreement (kappa = 0.71) with CTM. The clinical sensitivity and specificity of IS4 and CTM were 92.11% (35/38), 82.61% (76/92), 84.21% (32/38) and 95.65% (88/92), respectively. The clinical sensitivity and specificity of IS4 were similar for both pulmonary [92.00% (23/25) and 76.92% (30/39), respectively] and extrapulmonary [92.31% (12/13) and 86.79% (46/53), respectively] specimens. Among AFS-negative cases, the clinical sensitivity and specificity remained 90.48% (19/21) and 83.91% (73/87), respectively, with culture as the gold standard. We concluded that IS4, a new primer/probe pair for TaqMan based qPCR assay, was developed, optimized, and validated for the sensitive and specific detection of TB among various specimen types. The performance was not compromised under AFS-negative conditions.
结核病（TB）在全球范围内造成了沉重的健康负担，尤其是在发展中国家。快速准确地诊断结核病的需求尚未得到满足，尤其是对于肺外标本或抗酸染色（AFS）阴性的标本。因此，开发一种新型的、敏感的和特异性的检测方法来诊断结核病是非常必要的。
我们基于插入序列 6110（IS6110）开发了 IS4 引物/探针。通过 BLAST 设计了 qPCR 检测方法来检测 IS6110 中的特定区域。我们评估和优化了 IS4 引物/探针浓度、qPCR 效率和各种 PCR 添加剂。我们还使用 34 种常见分离的微生物来评估分析特异性。此外，我们收集了 130 份临床标本，与 Cobas TaqMan MTB（CTM）检测试剂盒和培养进行比较，以评估其性能。IS4 的扩增效率在没有和有内参 DNA（噬菌体 Lambda）时分别为 99.61%和 102.61%。二甲基亚砜（DMSO）在激发最有效的扩增和最低检测限方面优于甘油或 BSA。在评估临床性能时，我们收集了各种标本类型。IS4 与 CTM 具有高度一致性（kappa=0.71）。IS4 和 CTM 的临床敏感性和特异性分别为 92.11%（35/38）、82.61%（76/92）、84.21%（32/38）和 95.65%（88/92）。IS4 对肺内[92.00%（23/25）和 76.92%（30/39）]和肺外[92.31%（12/13）和 86.79%（46/53）]标本的临床敏感性和特异性相似。在 AFS 阴性病例中，以培养为金标准，其临床敏感性和特异性分别为 90.48%（19/21）和 83.91%（73/87）。
我们得出结论，IS4 是一种新的 TaqMan 实时 qPCR 检测方法的引物/探针对，它具有敏感性和特异性，可以用于检测各种标本类型的结核病。在 AFS 阴性的情况下，其性能不受影响。
