Kahbazi Manijeh, Sarmadian Hossein, Ahmadi Azam, Didgar Farshideh, Sadrnia Maryam, Poolad Toktam, Arjomandzadegan Mohammad
Infectious Diseases Research Center (IDRC), Arak University of Medical Sciences, Arak, 3819693345, I.R. of Iran.
Department of Pediatrics, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
Sci Pharm. 2018 Apr 16;86(2):15. doi: 10.3390/scipharm86020015.
In clinical isolates of (MTB), resistance to pyrazinamide occurs by mutations in any positions of the gene (NC_000962.3) especially in nucleotides 359 and 374. In this study we examined the gene sequence in clinical isolates of MTB. Genomic DNA of 33 clinical isolates of MTB was extracted by the Chelex100 method. The polymerase chain reactions (PCR) were performed using specific primers for amplification of 744 bp amplicon comprising the coding sequences (CDS) of the gene. PCR products were sequenced by an automated sequencing Bioscience system. Additionally, semi Nested-allele specific (sNASP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were carried out for verification of probable mutations in nucleotides 359 and 374. Sequencing results showed that from 33 MTB clinical isolates, nine pyrazinamide-resistant isolates have mutations. Furthermore, no mutation was detected in 24 susceptible strains in the entire 561 bp of the gene. Moreover, new mutations of G→A at position 3 of the gene were identified in some of the resistant isolates. Results showed that the sNASP method could detect mutations in nucleotide 359 and 374 of the gene, but the PCR-RFLP method by the SacII enzyme could not detect these mutations. In conclusion, the identification of new mutations in the gene confirmed the probable occurrence of mutations in any nucleotides of the gene sequence in resistant isolates of MTB.
在结核分枝杆菌（MTB）的临床分离株中，对吡嗪酰胺的耐药性是由pncA基因（NC_000962.3）任何位置的突变引起的，尤其是核苷酸359和374处的突变。在本研究中，我们检测了MTB临床分离株中的pncA基因序列。采用Chelex100法提取33株MTB临床分离株的基因组DNA。使用特异性引物进行聚合酶链反应（PCR），以扩增包含pncA基因编码序列（CDS）的744 bp扩增子。PCR产物通过自动测序生物科学系统进行测序。此外，还采用半巢式等位基因特异性（sNASP）和聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法来验证核苷酸359和374处可能的突变。测序结果显示，在33株MTB临床分离株中，9株对吡嗪酰胺耐药的分离株存在突变。此外，在24株敏感菌株的pncA基因全长561 bp中未检测到突变。而且，在一些耐药分离株中鉴定出pncA基因第3位的新突变G→A。结果表明，sNASP方法可检测pncA基因核苷酸359和374处的突变，但SacII酶的PCR-RFLP方法无法检测到这些突变。总之，pncA基因新突变的鉴定证实了MTB耐药分离株中pncA基因序列任何核苷酸处可能发生突变。
