Analyzing the mutations of rpoB gene in Mycobacterium tuberculosis clinical isolates by probe melting analysis assay / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical performance of a probe melting analysis (PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB).</p><p><b>METHODS</b>The specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM), and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing.</p><p><b>RESULTS</b>Among 37 NTM strains, three strains showed drug resistant mutation signals. The PMA method could detect down to 30 bacteria per reaction. Sample analysis showed that 186 of 962 isolates were mutants, 751 isolates were wild type and 25 isolates failed to give amplification signals. Among the mutant samples detected, 112 samples from November 2009 to April 2010 were further analyzed by sequencing, as well as 200 wild-type samples. The results showed a complete agreement with the PMA assay except for 5 samples failed in sequence analysis.</p><p><b>CONCLUSION</b>The PMA assay is rapid, accurate and easy-to-use, and thus can be used for detection of rifampin-resistant in clinical isolate samples.</p>

The etiological identification of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city, 2011 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To identify the etiology of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city from 11 to 17 May, 2011.</p><p><b>METHODS</b>A total of ten patients' throat swabs, anal swabs and cerebrospinal fluid were collected and detected by RT-PCR for pan-enterovirus. The samples containing detectable pan-enterovirus were tested by PCR with genotype-specific general primers located in VP1 region of enterovirus genotype A, B and C (HEV-A, B and C). The PCR products of VP1 segment were purified and sequenced, and phylogenetic analysis was performed. Meanwhile, the pathogens in those samples were isolated in Vero cell culture. Homologous analysis of VP1 sequences were carried out for the cultured virus samples and the original clinical samples to identify the outbreak etiology.</p><p><b>RESULTS</b>Among the ten cases, seven cases were positive for pan-enterovirus nucleic acid. When tested by genotype-specific PCR, the throat and anal swab samples from those 7 patients were positive with HEV-B VP1 primers. Meanwhile, the HEV-B VP1 segments were sequenced and phylogenetic analyzed, which indicated the seven cases were all infected by enterovirus Echo 30. The sequences from those samples had homology of 95.3% - 97.1% with the epidemic strains in Zhejiang, 2004. Out of the seven cases, the sequences of XM2, XM3, XM4, XM8 throat swab samples and XM3, XM6 throat samples showed 99.4% - 100.0% homology which were different from the sequence of XM1, and the homology was 92.8% - 93.4%. Furthermore, the viruses were isolated using Vero cells from XM1, XM2, XM3, XM4 and XM8 throat swab samples, and the VP1 sequence showed more than 99.9% homology with the original specimens.</p><p><b>CONCLUSION</b>The local outbreak of aseptic encephalitis was caused by Echo 30 of enterovirus genotype B, and the epidemic strains may have different genetic background.</p>

Application study on multicolor combinational probe coding real-time PCR in detection of foodborne pathogens / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the detection limit of multicolor combinational probe coding real-time PCR (MCPC-PCR) in detection of Salmonella and Staphylococcus aureus suspended in the food samples, and to apply MCPC-PCR to detect the samples of food poisoning.</p><p><b>METHODS</b>Series concentration of bacterium suspension (10(1) - 10(9) CFU/ml) was prepared by using 22 simulated samples including fresh meat and cakes and then MCPC-PCR was applied to detect Salmonella and Staphylococcus aureus in 22 samples. Enrichment broth of 101 frozen samples and 5 early patients' anal swabs in food poisoning cases were detected after the DNA samples were extracted.</p><p><b>RESULTS</b>The limits of MCPC-PCR assay in detecting Salmonella and Staphylococcus aureus were about 10(2) copies/test; 101 frozen enrichment broth of samples in food poisoning cases were detected by MCPC-PCR assay, of 23 positive samples, 18 were confirmed by bacteriology techniques; 96 samples detected by MCPC-PCR and bacteriology techniques had the same results, and the coincidence rate was 95.05%. Anal swabs, collected from 5 of early patients in a food poisoning case gave a clue to be Vibrio parahaemolyticus by MCPC-PCR assay and then were perfectly consistent with bacteriology assay.</p><p><b>CONCLUSION</b>As a method of high sensitivity and good specificity, MCPC-PCR assay can quickly and conveniently detect multiple pathogens existing in food samples, therefore we recommend it to be used in rapidly screening or simultaneous detection of food-borne diseases.</p>