Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
J Microbiol Methods. 2021 Apr;183:106184. doi: 10.1016/j.mimet.2021.106184. Epub 2021 Mar 1.
Diseases caused by typhoidal and non-typhoidal Salmonella remain a considerable threat to both developed and developing countries. Based on the clinical symptoms and serological tests, it is sometimes difficult to differentiate the Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) from serovar Typhi (S. enterica serovar Typhi). In this study, we developed a quadruplex real-time polymerase chain reaction (PCR) assay with an internal amplification control (IAC), to simultaneously differentiate S. enterica serovar Paratyphi A from serovar Typhi and to detect other Salmonella serovars which cause salmonellosis in humans. This assay was evaluated on 155 salmonellae and non-salmonellae strains and demonstrated 100% specificity in species differentiation. Inclusion of an IAC did not affect the efficiency of the assay. Further evaluation using a blind test on spiked stool, blood and food specimens showed that the detection limit was at 10 -10 CFU/mL (or g) and a high PCR efficiency with different targets (R > 0.99), except for S. enterica serovar Paratyphi A in blood. This assay has been applied to clinical specimens to detect the causative agents of gastrointestinal infections and has successfully identified 6 salmonellosis patients from the 50 diarrhoea patients. The quadruplex real-time PCR developed in this study could enhance the detection and differentiation of salmonellae. This assay could be applied to stools, blood and food based on the notable performance in the simulation tests and field evaluation.
伤寒和非伤寒沙门氏菌引起的疾病仍然是发达国家和发展中国家的重大威胁。根据临床症状和血清学检测，有时很难将甲型副伤寒沙门氏菌（S. enterica serovar Paratyphi A，S. enterica serovar Paratyphi A）与伤寒沙门氏菌（S. enterica serovar Typhi，S. enterica serovar Typhi）区分开来。在本研究中，我们开发了一种四重实时聚合酶链反应（PCR）检测方法，带有内部扩增对照（IAC），可同时区分甲型副伤寒沙门氏菌和伤寒沙门氏菌，并检测引起人类沙门氏菌病的其他沙门氏菌血清型。该检测方法在 155 株沙门氏菌和非沙门氏菌菌株上进行了评估，在物种分化方面表现出 100%的特异性。包含 IAC 不会影响检测方法的效率。进一步使用粪便、血液和食物标本的盲法检测进行评估表明，检测限为 10 -10 CFU/mL（或 g），不同靶标（R>0.99）的 PCR 效率高，除血液中的甲型副伤寒沙门氏菌外。该检测方法已应用于临床标本，以检测胃肠道感染的病原体，并成功从 50 例腹泻患者中鉴定出 6 例沙门氏菌病患者。本研究中开发的四重实时 PCR 可增强沙门氏菌的检测和鉴定。该检测方法可根据模拟测试和现场评估中的显著性能应用于粪便、血液和食物。
