Hanchaina Rattanavinan, Lulitanond Viraphong, Namwat Wises, Faksri Kiatichai
Southeast Asian J Trop Med Public Health. 2014 Mar;45(2):365-74.
Large sequence polymorphisms (LSPs) or regions of differences (RDs) are molecular epidemiological and evolutionary markers used to classify Mycobacterium tuberculosis (MTB) into East Asian (Beijing), Indo-Oceanic (IO), Euro-American (EuA) and East African Indian (EAI) lineages. The most used method is separate PCR and sequencing for each RD. We developed a single-tube multiplex PCR using four primer pairs specific to the four MTB lineages and a primer pair for species-specific RD9 with genomic DNA extracted from isolated colonies. The single-tube multiplex PCR produced lineage-specific amplicon patterns capable of differentiating the four MTB lineages. Sensitivity and specificity of the assay were 100% when differentiating MTB lineages from other species and strains of bacteria. The limit of detection of genomic MTB DNA was 12.5 ng. This single-tube multiplex PCR method offers a simple, rapid and reliable method for classification of MTB lineages based on LSPs.
大序列多态性（LSPs）或差异区域（RDs）是用于将结核分枝杆菌（MTB）分为东亚（北京）、印度洋（IO）、欧美（EuA）和东非印度（EAI）谱系的分子流行病学和进化标记。最常用的方法是对每个RD进行单独的PCR和测序。我们开发了一种单管多重PCR，使用针对四个MTB谱系的四对引物以及针对物种特异性RD9的一对引物，从分离菌落中提取基因组DNA。单管多重PCR产生了能够区分四个MTB谱系的谱系特异性扩增子模式。当区分MTB谱系与其他细菌物种和菌株时，该检测方法的灵敏度和特异性均为100%。基因组MTB DNA的检测限为12.5 ng。这种单管多重PCR方法为基于LSPs的MTB谱系分类提供了一种简单、快速且可靠的方法。
