Priyadarshini P, Tiwari K, Das A, Kumar D, Mishra M N, Desikan P, Nath G
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
Department of Microbiology, Bhopal Memorial Hospital and Research Centre, Bhopal, India.
Int J Tuberc Lung Dis. 2017 Feb 1;21(2):214-217. doi: 10.5588/ijtld.16.0343.
To evaluate the sensitivity and specificity of a new nested set of primers designed for the detection of Mycobacterium tuberculosis complex targeting a highly conserved heat shock protein gene (hsp65).
The nested primers were designed using multiple sequence alignment assuming the nucleotide sequence of the M. tuberculosis H37Rv hsp65 genome as base. Multidrug-resistant Mycobacterium species along with other non-mycobacterial and fungal species were included to evaluate the specificity of M. tuberculosis hsp65 gene-specific primers.
The sensitivity of the primers was determined using serial 10-fold dilutions, and was 100% as shown by the bands in the case of M. tuberculosis complex. None of the other non M. tuberculosis complex bacterial and fungal species yielded any band on nested polymerase chain reaction (PCR). The first round of amplification could amplify 0.3 ng of the template DNA, while nested PCR could detect 0.3 pg.
The present hsp65-specific primers have been observed to be sensitive, specific and cost-effective, without requiring interpretation of biochemical tests, real-time PCR, sequencing or high-performance liquid chromatography. These primer sets do not have the drawbacks associated with those protocols that target insertion sequence 6110, 16S rDNA, rpoB, recA and MPT 64.
评估一组新的巢式引物的敏感性和特异性，该引物设计用于检测结核分枝杆菌复合群，靶向一个高度保守的热休克蛋白基因（hsp65）。
以结核分枝杆菌H37Rv hsp65基因组的核苷酸序列为基础，通过多序列比对设计巢式引物。纳入耐多药分枝杆菌物种以及其他非分枝杆菌和真菌物种，以评估结核分枝杆菌hsp65基因特异性引物的特异性。
使用系列10倍稀释法测定引物的敏感性，对于结核分枝杆菌复合群，条带显示敏感性为100%。其他非结核分枝杆菌复合群的细菌和真菌物种在巢式聚合酶链反应（PCR）中均未产生任何条带。第一轮扩增可扩增0.3 ng模板DNA，而巢式PCR可检测到0.3 pg。
已观察到目前的hsp65特异性引物灵敏、特异且具有成本效益，无需对生化试验、实时PCR、测序或高效液相色谱进行解读。这些引物组没有与靶向插入序列6110、16S rDNA、rpoB recA和MPT 64的那些方案相关的缺点。
