Francisella tularensis is a fastidious, Gram-negative coccobacillus and is the causative agent of tularemia. To assess viability yet overcome lengthy incubation periods, a culture-based PCR method was used to detect early growth of the lowest possible number of F. tularensis cells. This method utilized a previously developed enhanced F. tularensis growth medium and is based on the change in PCR cycle threshold at the start and end of each incubation. To test method robustness, a virulent Type A1 (Schu4) and B (IN99) strain and the avirulent Live Vaccine Strain (LVS) were incubated with inactivated target cells, humic acid, drinking and well water, and test dust at targeted starting concentrations of 1, 10, and 100 CFU mL− 1 (low, mid, and high, respectively). After 48 h, LVS growth was detected at all targeted concentrations in the presence of 106 inactivated LVS cells; while Schu4 and IN99 growth was detected in the presence of 104 Schu4 or IN99 inactivated cells at the mid and high targets. Early detection of F. tularensis growth was strain and concentration dependent in the presence of fast-growing well water and test dust organisms. In contrast, growth was detected at each targeted concentration by 24 h in humic acid and drinking water for all strains. Results indicated that the culture-based PCR assay is quick, sensitive, and specific while still utilizing growth as a measure of pathogen viability. This method can circumvent lengthy incubations required for Francisella identification, especially when swift answers are needed during epidemiological investigations, remediation efforts, and decontamination verification.

中文翻译：

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通过基于文化的PCR早期检测环境基质中的土拉弗朗西斯菌。

图拉弗朗西斯菌是一种挑剔的革兰氏阴性球菌，是图拉菌血症的病原体。为了评估生存力并克服了漫长的潜伏期，使用了一种基于培养物的PCR方法来检测尽可能少数量的F. tularensis细胞的早期生长。该方法利用了以前开发的增强的土拉弗朗西斯菌生长培养基，并且基于每次孵育开始和结束时PCR循环阈值的变化。为了测试方法的稳健性，将有毒的A1型（Schu4）和B型（IN99）菌株以及无毒的活疫苗株（LVS）与灭活的靶细胞，腐殖酸，饮用水和井水一起温育，并在目标起始浓度为1、10和100 CFU mL-1（分别为低，中和高）。48小时后，在106个灭活的LVS细胞存在的情况下，在所有目标浓度下均检测到LVS生长。而在中高目标位处有104个Schu4或IN99失活的细胞存在时，检测到Schu4和IN99的生长。在快速生长的井水和试验粉尘生物的存在下，土拉弗朗西斯菌生长的早期检测取决于菌株和浓度。相比之下，对于所有菌株，在腐殖酸和饮用水中检测到在每个目标浓度下24 h都有生长。结果表明，基于培养物的PCR检测快速，灵敏且特异，同时仍利用生长来衡量病原体的生存能力。这种方法可以避免进行弗朗西斯菌鉴定所需的长时间孵育，尤其是在流行病学调查，补救措施，