基于物种特异性PCR技术检测鼻疽诺卡菌和巴西诺卡菌的初步研究

Preliminary detection of Nocardia farcinica and Nocardia brasiliensis through species-specific PCR

目的建立一种可以鉴别鼻疽诺卡菌和巴西诺卡菌的双重物种特异性PCR(species-specific Polymerase Chain Reaction,SS-PCR)诊断方法。方法基于鼻疽诺卡菌和巴西诺卡菌物种特异基因NFA_41530和O3I_RS18895分别设计特异性引物,通过优化反应条件和体系,建立快速准确的双重SS-PCR。对95株鼻疽诺卡菌、13株巴西诺卡菌和42株非目标菌株进行扩增,分析该方法的特异性和灵敏度,并通过模拟痰样本验证方法的准确性,预估其在临床诊断的价值。结果鼻疽诺卡菌和巴西诺卡菌质检分别扩增出一条清晰且单一的目标条带,其余42株非目标菌株均未扩增,表明该方法具有很好的特异性。该方法对鼻疽诺卡菌、巴西诺卡菌的检测下限分别为1.3×10^(4)copies/μL、2.4×10^(4)copies/μL,灵敏度较高。使用20个健康人的痰液模拟痰样本,检测符合率为100%。结论基于NFA_41530和O3I_RS18895基因建立的双重SS-PCR方法特异性强、灵敏度较高,是一种可以同时鉴定鼻疽诺卡菌和巴西诺卡菌的简便、快速、可靠、经济的方法。

This study was aimed at establishing a duplex species-specific polymerase chain reaction(SS-PCR)method for the identification of Nocardia farcinica and N.brasiliensis.Specific primers were designed on the basis of the species-specific genes NFA_41530 and O3I_RS18895 of N.farcinica and N.brasiliensis,respectively.Rapid and accurate duplex SS-PCR was established by optimization of the reaction conditions and system.Then 95 strains of N.farcinica,13 strains of N.brasiliensis and 42 strains of non-target strains were amplified.The specificity and sensitivity of the method were analyzed,and its clinical diagnostic value was estimated through the identification of simulated sputum samples.A single clear target band was amplified for N.farcinica and for N.brasiliensis,and the other 42 non-target strains were not amplified,thus indicating that the method had good specificity.The detection limit for N.farcinica and N.brasiliensis was 1.3×10^(4)copies/μL and 2.4×10^(4)copies/μL respectively,with high sensitivity.Twenty sputum samples from healthy people were used assimulated sputum samples,and the detection coincidence rate was 100%.The SS-PCR method based on NFA_41530 and O3I_RS18895 genes has high specificity and sensitivity,and is a simple,rapid,reliable and economical method for the simultaneous identification of N.farcinica and N.brasiliensis.