ABSTRACT
<p><b>OBJECTIVE</b>To develop a high-throughput rapid method for Vibrio (V.) cholerae molecular typing based on Melting Curve-based Multilocus Melt Typing (McMLMT).</p><p><b>METHODS</b>Seven housekeeping genes of V.cholerae were screened out, and for each gene, the specific primers were designed for correspondent genes as well as 4 probes covering polymorphism loci of sequences. After optimizing all parameters, a method of melting-curve analysis following asymmetric PCR was established with dual-fluorescent-reporter in two reaction tubes for each gene. A set of 28 Tm-values was obtained for each strain and then translated into a set of code of allelic genes, standing for the strain's McMLMT type (MT). Meanwhile, sequences of the 7-locus polymorphism were typed according to the method of MLST. To evaluate the efficiency and reliability of McMLMT, the data were compared with that of sequence-typing and PFGE using BioNumerics software.</p><p><b>RESULTS</b>McMLMT method was established and refined for rapid typing of V. cholerae that a dozen of strains can be finished testing in a 3-hours PCR running using 96-well plates. 108 strains were analyzed and 28-Tm-values could be grouped and encoded according to 7 housekeeping gene to obtain the code set of allelic genes, and classified into 18 types (D = 0.723 3). Sequences of the 7 genes' polymorphism areas were directly clustered into the same 18 types with reference to MLST method. 46 of the strains, each represented a different PFGE type, could be classified into 13 types (D = 0.614 5) with McMLMT method and A- K groups at 85% similarity (D = 0.858 9) with PFGE method.</p><p><b>CONCLUSION</b>McMLMT method is a rapid high-throughput molecular typing method for batches of strains with a resolution equal to MLST method and comparable to PFGE group.</p>
Subject(s)
Multilocus Sequence Typing , Polymerase Chain Reaction , Vibrio choleraeABSTRACT
Objective To evaluate the potential use of a probe melting analysis (PMA) assay in detecting the embB mutations which confer resistance against ethambutol in Mycobacterium tuberculosis. Methods The analysis sensitivity and specificity of PMA were investigated by detecting a serially diluted H37 Rv DNA and a reference panel from National Institute for the Control of Pharmaceutical and Biological Product. Six hundred and thirteen sputum samples were collected from the Xiamen Center for Disease Control and Prevention, Xiamen First Hospital and Center for Zhangzhou Disease Control and Prevention from September 2009 to April 2010. The PMA assay was then evaluated by detecting 613 clinical isolates and the results were compared with the sequencing results. Results The PMA assay could specifically detect Mycobacterium tuberculosis and had a limit of detection of 3 copies per reaction. The assay results with 613 clinical isolates showed that PMA gave a 100% concordance with sequencing in the 583 qualified samples, among which 34 were mutations at embB 306,23 at embB 378-380, 3 at embB 406 and 3 at embB 497. Conclusions PMA assay is a sensitive and specific method enabling efficient detection of common embB mutations causing ethambutol-resistance. The rapidness of this method together with its reliability would facilitate its use in routine testing.
ABSTRACT
The purpose of the present study was to investigate Norovirus infection status among patients with virus diarrhea in Xiamen city and provide evidence for exploring the prevalence characteristics and constituting appropriate control strategy.From April 2007 to July 2008,323 fecal samples of virus diarrhea cases collected from 3 surveillance hospitals in Xiamen were detected for antigen and RNA by ELISA and Real-Time RT-PCR respectively.The RdPd genes from some samples were furtherly amplified and sequenced for genogroup identification when the Real-Time RT-PCR detection results were positive.In the 323 fecal specimens,68 (21.05%) were positive for Norovirus antigen by ELISA and 107 (31.13%) were positive for Norovirus RNA by Real-Time RT-PCR.The overall positive prevalence rate of Norovirus in Xiamen was 38.08%.107 positive specimens were detected by Real-Time RT-PCR and results showed that 80 strains were Norovirus GGⅡ(74.77%),2 strains were GGⅠ(1.87%) and 25 strains (23.36%)were unidentified.It's indicated that Norovirus infection in Xiamen district was mainly caused by Norovirus GGⅡ,and Norovirus was also the main cause for virus diarrhea.