快速检测洋葱伯克霍尔德菌巢式荧光定量PCR法建立  

Establishment of nested fluorescent quantitative PCR for rapid detection of Burkholderia cepacia

作　　者：林丽英[1] 邓穗燕[1] 郭旭光[1] LIN Liying;DENG Suiyan;GUO Xuguang(Department of Clinical Laboratory,the Third Affiliated Hospital of Guangzhou Medical University,Guang⁃zhou Guangdong,China,510150)

机构地区：[1]广州医科大学附属第三医院检验科,广东广州510150

出　　处：《分子诊断与治疗杂志》2020年第4期437-440,458,共5页Journal of Molecular Diagnostics and Therapy

基　　金：广州市医药卫生科技项目(20161A010082)。

摘　　要：目的建立能直接快速应用于临床检测洋葱伯克霍尔德菌的巢式荧光定量PCR法。方法根据洋葱伯克霍尔德菌recA基因序列设计用于普通PCR扩增的外引物,同时设计用于荧光定量PCR的内引物。分别以洋葱伯克霍尔德菌等细菌的标准株为模板,以外引物进行第一轮PCR扩增;所得PCR产物为模板,以内引物进行染料法荧光定量PCR检测recA基因表达水平,评估巢式荧光定量PCR法的特异性。同时将洋葱伯克霍尔德菌的菌液和基因组DNA进行浓度梯度稀释,继续进行巢式荧光定量PCR,确定此方法的灵敏度。最后用巢式荧光定量PCR法和Vitek 2 Compact仪器分别鉴定35例临床标本,比较两者的检出能力。结果巢式荧光定量PCR法只针对洋葱伯克霍尔德菌有扩增曲线,而对其他菌株无检测信号。同时,巢式荧光定量PCR法的检测下限是1×10^1 CFU/mL的菌液和1×10^⁃5 ng/μL的基因组DNA,具有较高的灵敏度。巢式荧光定量PCR法能有效检测出14例临床标本存在洋葱伯克霍尔德菌感染(检出率为40.0%),而Vitek 2 Compact仪器仅检测出10例(检出率为28.6%),其中4例无法识别。结论巢式荧光定量PCR法具有有效、快速和直接应用于临床检测的优点,并且特异、灵敏和准确等特点,值得在临床推广。Objective To establish a nested fluorescent quantitative PCR method for the detection of Burkholderia cepacia(B.cepacia).Methods According to the sequence of recA gene,the external prim⁃ers for PCR amplification and the internal primers for fluorescent quantitative PCR were designed.Using the reference strains of B.cepacia and other bacteria as templates,the first round of PCR amplification was carried out with external primers;the PCR products were used as templates,and the expression level of recA gene was detected by SYBR Green dyebased fluorescent quantitative PCR with internal primers to evaluate the speci⁃ficity of nested fluorescent quantitative PCR.At the same time,the bacterial solution and genomic DNA of B.cepacia were diluted by concentration gradient,and the nested fluorescent quantitative PCR was carried out to determine its sensitivity.Finally,20 clinical specimens were identified by nested fluorescent quantitative PCR and VITEK 2 compact respectively,and their detection ability was compared.Results Nested fluorescent quantitative PCR only had amplification curve for B.cepacia,but no detection signal for other strains.At the same time,the detection limit of nested fluorescent quantitative PCR was 1×10^1 CFU/mL bacterial solution and 1×10^⁃5 ng/μL genomic DNA,which had high sensitivity.The nested fluorescent quantitative PCR effectively detected B.cepacia infection in 14 clinical samples(detection rate=40.0%),but only 10 cases were detected by VITEK 2 compact instrument(detection rate=28.6%),among which 4 cases could not be identified,indicating that the nested fluorescent quantitative PCR had more accurate detection performance.Conclusion Nested fluorescent quantitative PCR is an effective,rapid and direct method for clinical detection of Burkholde⁃ria cepacian.

关 键 词：洋葱伯克霍尔德菌 RECA基因 巢式荧光定量PCR 临床检测 

分 类 号：R446.5[医药卫生—诊断学]