ABSTRACT
Objective To establish a method for the identification of Vibrio parahaemolyticus based on Taqman-fluorescence probe quantitative PCR method targeting toxR gene.Methods Taking the standard strain of Vibrio parahaemolyticus (VPJS421) and other ommon pathogenic bacteria'standard strain as the research object,using the bio-software to design specific PCR primers and Taqman probe of Vibrio parahaemolyticus toxR gene and detected by fluorescence quantitative PCR instrument.Results ①The designed primers could amplify specific bands.②The amplification efficiency of the 0.5 μl probe in the amplification system was better than that of the 1.0μl probe.③The detection sensitivity of toxR gene of Vibrio parahaemolyticus by Taqman fluorescence quantitative PCR was 10-1 mg/L.④The detection method did not show positive amplification in detection of Enterococcus f aecalis,Staphylococcus aureus,Saprophytic staphylococcus,Enterobacter hormaechei,Pseudomonas aeruginosa,Escheri ch ia coli,Vibrio al ginol yticus,Vibrio vulnficus,Vibrio metschnikovii and Wbrio furnissii 10 other common pathogenic bacteria.The specificity was 100％.Conlusion The fluorescence quantitative PCR method for the identification of Vibrio parahaemolyticus was successfully established.The method was sensitivty and specificity,and it is suitable for rapid detection of Wbrio parahaemolyticus and has a good application value.
ABSTRACT
Objective To establish the method of identifying MRSA with Taqman-fluorescence quantitative PCR basing on mecA/nuc/fem B three gene combined detecting.Methods Taking the coagulase positive MRSA,which isolated from the clinical samples and confirmed by VITEK 2 compact microbial analyzer,as the research obj ect,designed mecA/nuc/fem B specific PCR primers and Taqman fluorescent probe by bio-software PrimerPremier 5 and Designer Beacon 7,FAM,HEX and ROX markers were used to label the fluorescent probe at 5’,and the end of 3’was labeled with BHQ1,detected by fluo-rescence quantitative PCR instrment.Results ①1 g/dl gel electrophoresis results showed that the primer’s specificity of mec A/nuc/fem B were good,and molecular weight of the amplification band consistent with the expected molecular weight and no non-specific amplification band.②Three genes were obtained specific amplification in a single tube single channel and single tube multiple channel detection in PCR,and the three gene amplification effect in a single tube single tube single chan-nel and multichannel PCR similar.Conclusion Successfully established a method of multi channel Taqman-probe fluores-cence quantitative PCR identification of MRSA,mec A/nuc/fem B combined detection can effectively differentiate coagulase negative and positive MRSA,improve the accuracy of identification.
ABSTRACT
Objective To establish a method for the rapid detection of hepatitis E virus (HEV)from serum samples based on fluorescence quantitative PCR.Methods (1 )One-hundred HEV sequences including our country popular three major genotypes were obtained from the GeneBank with the Vector NTI software.The proper sequence was selected to design and synthesize the primers of the fluorescence quantitation and the Taqman probe.(2)The amplification region PCR fragment was transcribed in vitro to synthesize cRNA standard,at the same time the trace serum virus lysate was introduced into a universal real-time TaqMan PCR assay.(3)10 clinical serum samples were collected from the patients with clinical hepatitis E and detected by using the established method for further verifying this method.Results This detection technique could effectively detect the serum samples in the pa-tients with genotype I and genotype IV hepatitis E positive,while the serum detection in the patients with other virus infectious dis-eases had the negative results,which verified that this RT-PCR detection technique had higher specificity and good reliability.The detection results from 10 clinical serum samples further verified that this method was rapid,convenient and sensitive with good re-peatability.Conclusion A fluorescence quantitative RT-PCR detection technique suitable for detecting main genotypes of HEV in China population is established,which can meet the demand of early and rapid diagnosis for HEV.