Establishment Of The Multiple PCR And Taqman Real-time PCR Method For Detecting Nocardia

Nocardia is an opportunistic pathogen,it is caused nocardiosis that can occurmany race,occupation,age,it main causes lung,skin infections and acute or chronicpyogenic granulomatous diseases,sometimes invasive brain,abscess formation,canserious or even death of the patient.Researchers through a lot of research foundthat,nocardia infections are increasing year by year.Nocardia conventionalidentification method is isolated culture,it will take a lot of time andmanpower,usually need one or three weeks to complete.With the development ofmolecular biology,PCR detection for Nocardia have also been developed.MultiplePCR and Taqman probe real-time PCR methods both have the advantages of goodspecificity,high sensitivity,high degree of automation,are widely used inmicrobiological testing.Detect nocardia by these two methods,they can provided areliable reference standards for clinical rapid and accurate diagnosis of Nocardia.ObjectiveThe study established the multiple PCR and Taqman real-time PCR method fordetecting Nocardia, provided a reliable reference standards for clinical rapid andaccurate diagnosis of Nocardia.Methods1. Used multiplex PCR method, three primers were designed for detectingrpoB,SecA1,16SrRNA genes of Nocardia, optimized the reaction conditions andreaction system, then the sensitivity and specificity of the multiple PCR methodwere tested by44Nocardia standard strains，44clinical isolates and7referencestrains were tested under the same reaction system and condition.2. Designed primers and probes of rpoB gene for Nocardia, then determineddetection limit of the method by made standard curve, verified practical application of the experiment through sputum simulative specimens.Results1. The results demonstrated that,2strains of Nocardia (DSM43003,CDC51)wereamplified by using a single pair primer, the single bands obtained were consistentwith target fragment for the same length. Then the target genes were verified bysequencing and BLAST, the specificities of three primers met the test requirement.With the established multiple PCR method,3segment were confirmed in43(97.7%)of44Nocardia standard strains and42(95.5%) of isolates strains, however, therewere no band obtained in7control strains, the specificity met the test requirement,and the detection limits for DNA template were1x10-4ng.2. Established the real-time PCR method for rapid detection of Nocardia insimulated clinical specimens,standard curve showed that23copy Nocardia perreaction could be detected, the lowest detection limit of the method were2.0×102cfuperml of sputum specimens. In specific experiments, the results of detection werepositive for samples of Nocardia gene, but negative for genome of other bacteria.Conclusions1. It concluded that the multiple PCR and Taqman probe Real-time PCRmethod are fast, specific, rigorous, sensitive,they can discriminate Nocardia in12hours.And can be used for identifying Nocardia strains in clinical.2. Comparative Multiple PCR and Taqman probe Real-time PCRmethod,Multiple PCR is cheap,and experimental operation is simple and easy tograsp;but Taqman probe Real-time PCR method can discriminate Nocardia inabsolute quantification,more sensitivity,2.0×102cfu/ml of Nocardia colonies can bediscovered.