A fast and highly sensitive blood culture PCR method for clinical detection of Salmonella enterica serovar Typhi
A fast and highly sensitive blood culture PCR method for clinical detection of Salmonella enterica serovar Typhi
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DOI：
10.1186/1476-0711-9-14
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发表时间：
2010-01-01
影响因子：
5.7
通讯作者：
Pollard, Andrew J.
中科院分区：
文献类型：
--
作者：
Zhou, Liqing;Pollard, Andrew J.
背景：伤寒沙门氏菌每年估计导致2100万新的伤寒病例和216，000人死亡。血培养是目前诊断伤寒的金标准，但它很耗时，需要几天的时间来分离和鉴定病原体。此时再开始适当的抗生素治疗为时已晚。血清学检测的敏感性和特异性很低，在流行区无实用价值。由于疾病的早期诊断和及时治疗对于最佳管理至关重要，特别是在儿童中，因此迫切需要一种快速敏感的伤寒检测方法。虽然PCR是敏感和快速的，但初步研究表明与血培养相似的敏感性和较低的特异性。我们开发了一种快速，高灵敏度的血液培养PCR方法检测伤寒沙门氏菌，允许当天开始治疗后，准确诊断typhoid.Methods：牛胆汁胰蛋白胨大豆肉汤进行了优化的血液培养，它允许完全溶解的血细胞释放细胞内的细菌，而不抑制伤寒沙门氏菌的生长。使用优化的肉汤，人工血液样品中的伤寒沙门氏菌在血液培养物中富集，然后通过靶向伤寒沙门氏菌fliC-d基因的PCR检测。实验证明，2.4%牛胆汁在血培养中不仅在1.5小时内完全溶解血细胞，使细胞内细菌释放，而且对伤寒沙门氏菌的生长无抑制作用。伤寒沙门氏菌在含2.4%胰蛋白胨大豆肉汤中富集3小时，牛胆汁可使细菌数从0.75CFU/ml血液（与临床伤寒样品相似）增加到常规PCR可检测的水平。结论：全血培养PCR检测方法在速度和灵敏度上均优于传统的血培养和PCR检测方法，具有上级优势。它在临床诊断中的使用可能允许早期检测致病微生物，并促进伤寒患者的及时治疗。
Background: Salmonella Typhi causes an estimated 21 million new cases of typhoid fever and 216,000 deaths every year. Blood culture is currently the gold standard for diagnosis of typhoid fever, but it is time-consuming and takes several days for isolation and identification of causative organisms. It is then too late to initiate proper antibiotic therapy. Serological tests have very low sensitivity and specificity, and no practical value in endemic areas. As early diagnosis of the disease and prompt treatment are essential for optimal management, especially in children, a rapid sensitive detection method for typhoid fever is urgently needed. Although PCR is sensitive and rapid, initial research indicated similar sensitivity to blood culture and lower specificity. We developed a fast and highly sensitive blood culture PCR method for detection of Salmonella Typhi, allowing same-day initiation of treatment after accurate diagnosis of typhoid.Methods: An ox bile tryptone soy broth was optimized for blood culture, which allows the complete lysis of blood cells to release intracellular bacteria without inhibiting the growth of Salmonella Typhi. Using the optimised broth Salmonella Typhi bacteria in artificial blood samples were enriched in blood culture and then detected by a PCR targeting the fliC-d gene of Salmonella Typhi.Results: Tests demonstrated that 2.4% ox bile in blood culture not only lyzes blood cells completely within 1.5 hours so that the intracellular bacteria could be released, but also has no inhibiting effect on the growth of Salmonella Typhi.Three hour enrichment of Salmonella Typhi in tryptone soya broth containing 2.4% ox bile could increase the bacterial number from 0.75 CFU per millilitre of blood which is similar to clinical typhoid samples to the level which regular PCR can detect. The whole blood culture PCR assay takes less than 8 hours to complete rather than several days for conventional blood culture.Conclusions: This novel blood culture PCR method is superior in speed and sensitivity to both conventional blood culture and PCR assays. Its use in clinical diagnosis may allow early detection of the causative organism and facilitate initiation of prompt treatment among patients with typhoid fever.