Shevtsov Alexandr, Dauletov Ayan, Izbanova Uinkul, Kairzhanova Alma, Tursunbay Nailya, Kiyan Vladimir, Vergnaud Gilles
National Center for Biotechnology, Astana 010000, Kazakhstan.
Aikimbayev's National Scientific Center for Especially Dangerous Infections, Almaty 050000, Kazakhstan.
Microorganisms. 2024 Nov 16;12(11):2345. doi: 10.3390/microorganisms12112345.
Tularemia is an acute infectious disease classified as a natural focal infection, requiring continuous monitoring of both human and animal morbidity, as well as tracking of pathogen circulation in natural reservoirs and vectors. These efforts are essential for a comprehensive prevention and containment strategy. The causative agent, , comprises three subspecies-, , and -which differ in their geographic distribution and virulence. The ability to directly detect the pathogen and differentiate between subspecies has enhanced diagnostics and allowed a more accurate identification of circulation areas. Real-time PCR protocols for identification of subspecies and have been developed, utilizing specific primers and probes that target unique genomic regions. In this study, we present the development of a new real-time PCR assay for the detection of spp. and differentiation of subsp. . The specificity of the assay was tested on DNA from 86 bacterial species across 31 families unrelated to spp., as well as on DNA collections of subsp. and subsp. . The limit of detection (LOD95%) for real-time PCR in detecting spp. was 0.297 fg (0.145 genomic equivalents, GE) for DNA and 0.733 fg (0.358 GE) for DNA. The LOD95% for subspecies differential identification of was 8.156 fg (3.979, GE). The high sensitivity and specificity of these developed protocols enable direct detection of pathogens in biological and environmental samples, thereby improving the efficiency of tularemia surveillance in Kazakhstan.
土拉菌病是一种急性传染病，属于自然疫源性感染，需要持续监测人类和动物的发病率，以及追踪病原体在自然宿主和传播媒介中的循环情况。这些工作对于全面的预防和控制策略至关重要。病原体弗朗西斯菌属包含三个亚种，即土拉热弗朗西斯菌、新凶手弗朗西斯菌和假土拉热弗朗西斯菌，它们在地理分布和毒力上存在差异。直接检测病原体并区分亚种的能力提高了诊断水平，并有助于更准确地确定病原体的传播区域。利用针对独特基因组区域的特异性引物和探针，已经开发出用于鉴定土拉热弗朗西斯菌亚种和新凶手弗朗西斯菌的实时荧光定量PCR方法。在本研究中，我们展示了一种用于检测弗朗西斯菌属细菌并区分土拉热弗朗西斯菌亚种的新型实时荧光定量PCR检测方法的开发过程。该检测方法的特异性在来自31个与弗朗西斯菌属无关的科的86种细菌的DNA上进行了测试，同时也在土拉热弗朗西斯菌亚种和新凶手弗朗西斯菌的DNA样本上进行了测试。实时荧光定量PCR检测弗朗西斯菌属细菌的检测限（LOD95%）对于土拉热弗朗西斯菌DNA为0.297 fg（0.145个基因组当量，GE），对于新凶手弗朗西斯菌DNA为0.733 fg（0.358 GE）。土拉热弗朗西斯菌亚种差异鉴定的LOD95%为8.156 fg（3.979 GE）。这些开发的方法具有高灵敏度和特异性，能够直接检测生物和环境样本中的病原体，从而提高哈萨克斯坦土拉菌病监测的效率。
