应用单一和双重荧光定量PCR法快速检测军团菌

Single and duplex fluorescence quantitative PCR for rapid detection of Legionella

目的 建立单一和双重荧光定量PCR方法分别和同时进行军团菌属及嗜肺军团菌的检测.方法 利用军团菌属16 S rRNA基因和嗜肺军团菌mip基因设计引物和探针,两条基因探针分别标记FAM和HEX,并将相关反应体系和条件进行优化.分别应用单一基因探针（单一荧光定量PCR）和双重基因探针（双重荧光定量PCR）对嗜肺军团菌、非嗜肺军团菌及非军团菌进行检测,并验证两种方法的特异度、敏感度.应用双重荧光定量PCR检测空调水样滤膜样品和DNA提取样品,比较两者结果的一致性.结果 针对军团菌属及嗜肺军团菌,应用荧光定量PCR,16 S rRNA基因和mip基因均能较好的检出,16S rRNA和mip的最低检出限分别为8和10个拷贝.经优化得到了最佳反应体系.单一荧光定量PCR方法所检的8株嗜肺军用菌及4株非嗜肺军团菌16 S rRNA基因均为阳性,嗜肺军团菌mip基因阳性,非嗜肺军团菌mip基因阴性.双重荧光定量PCR方法所检的23株嗜肺军团菌中有2株为假阴性,9株非嗜肺军团菌和非军团菌属中有1株为假阳性.49份空调水样滤膜直接检测和提取DNA后检测的结果一致,其中26份水样军团菌阳性,20份为嗜肺军团菌,6份为非嗜肺军团菌;1份弗朗西斯菌检测HEX阳性（假阳性）,占实际培养分离的1/26.结论 单一及双重荧光定量PCR法特异、快速、敏感,一次同时检测嗜肺与非嗜肺军团菌,满足对空调和环境水样军团菌监测的要求.

Objective To develop the single and duplex fluorescence quantitative PCR for detection of Legionella and Legionella pneumophila respectively.Methods Specific primers and probes were designed according to the 16 S rRNA gene of Legionella and mip gene of Legionella pneumophila.The probes were labeled with reporter genes, FAM （for 16 S rRNA） and HEX （for mip gene） respectively.Moreover, the reaction systems and conditions for PCR were optimized.Then the single gene probe （single fluorescence quantitative PCR） and duplex gene probe （duplex fluorescence quantitative PCR） were used to detect the pneumophila and non-Legionella pneumophila strains, as well as other non-Legionella, in order to validate the specificity and sensitivity of these two methods.In addition, duplex fluorescence quantitative PCR was employed to detect Legionella on filtering membrane and extracted Legionella DNA in water samples from air - conditioners, to compare the consistency between the both.Results The 16 S rRNA genes of Legionella and mip genes of Legionella pneumophila were well detected with fluorescence quantitative PCR,with the lowest detectable templates being 8 and 10 copies respectively.An ideal reaction system after optimization of conditions was established successfully.Single fluorescence quantitative PCR demonstrated positive expression of 16 S rRNA gene in all of 8 Legionella pneumophila and 4 nonLegionella pneumophila strains, and positive expression of mip gene in Legionella pneumophila but not in non- Legionella pneumophila strains.Duplex fluorescence quantitative PCR demonstrated 2 false negative strains among 23 Legionella pneumophila strains and 1 false positive strain among 9 nonLegionella pneumophila and non - Legionella strains.The results of DNA detection were consistent between 49 filtering membranes and extracted DNA in water samples from air-conditioners.There were 26 water samples tested positive for Legionella, including 20 for Legionella pneumophila and 6 for nonLegionella pneumophila strains.One Francisella strain was found to be HEX-positive （false positive） ,accounting for 1/26 of the cultures actually isolated.Conclusions The single and duplex fluorescence quantitative PCR are specific, rapid and sensitive methods that detect pneumophila and non- Legionella pneumophila strains at the same time.These tests may meet the need to detect Legionella in water samples from air-conditioners or natural environment.