套式PCR检测唾液中变形链球菌及远缘链球菌

Rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by nested polymerase chain reaction

目的 探索一种快速、简便地从人类唾液中同时检测变形链球菌和远缘链球菌的方法。方法 分别以变形链球菌 gtfI和远缘链球菌 gtfB基因设计两组成套引物 ,首先用套式PCR(二次PCR)检测变形链球菌和远缘链球菌标准株和临床株 ,然后用套式PCR直接从唾液中检测这两种细菌。结果 变链菌 (血清型c,e ,f)的标准株及临床株第 1次PCR扩增产物为 5 17bp ,第 2次扩增产物为 4 6 8bp ;远缘链球菌 (血清型d ,g)及道勒链球菌 (血清型h)的标准株及临床株第 1次PCR扩增产物为 712bp ,第 2次扩增产物为 6 6 3bp ;其他异种菌均不能扩增出产物 ,因此该PCR检测具有高度的特异性。细菌纯培养物及唾液PCR检测的敏感性分别是 :第 1次PCR为 10 5CFU ,第 2次PCR为10 3 CFU。结论 套式PCR能快速在人类唾液中同时检测变形链球菌和远缘链球菌。该检测方法有望运用于临床检测 。

Objective To establish a simple and rapidmethod to detect Streptococcus mutans and Streptococcus sobrinus simultaneously in human saliva Methods Chromosomal DNA from the bacteria was obtained by the extraction method with phenol chloroform A nested PCR method with two sets of primers specific for portions of the glucosyltransferase genes (gtfB of S mutans and gtfI of S sobrinus ), was optimized to detect S mutans and S sobrinus from standard strains,clinical strains and directly in human saliva Results The first process of nested PCR was capable of amplifying DNA fragments specific for these species from chromosomal DNA extracted from 10 5 CFU cells of standard and clinical strains, or from 1ml clinical saliva samples containing 10 5 CFU cells of either species A second process of nested PCR, using the first PCR product as a template with new internal primers to detect 10 3 CFU of either streptococcal species in 1ml saliva samples Conclusion Nested PCR could detect S mutans and S sobrinus rapidly andsimply in human saliva This finding would be important to studies of elucidating the role of these two streptococcal species in the etiology of dental caries