Development of a rapid molecular typing method for Vibrio cholerae using melting curve-based multilocus melt typing / 中华预防医学杂志

<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>

The analysis of the genotyping of plasmid-mediated AmpC β-lactamases produced by clinical strains of Escherichia coli and Klebsiella pneumoniae / 国际检验医学杂志

Objective To investigate the genotype and epidemiology of plasmid‐mediated AmpC β‐lactamases produced by the clinical strains of Escherichia coli and Klebsiella pneumoniae .Methods A total of 176 clinical nonrepetitive cefoxitin non‐sensitivity isolates of Escherichia coli and Klebsiella pneumoniae was collected from July 2011 to August 2012 .Polymerase chain reaction (PCR) for AmpC enzyme gene amplification and DNA sequencing were carried out for genotype of AmpC beta‐lactamases .Results The results of PCR showed that the positive rate of ampC of the 176 strains of Escherichia coli and Klebsiella pneumoniae AmpC was 18 .2% ,mainly DHA type ,counting for 59 .4% ,CIT counting for 37 .5% ,EBC counting for 3 .1% .The positive rate of ampC of Escherichia coli was 11 .4% ,mainly CIT type ,counting for 77 .8% ,the positive rates of DHA type and EBC type both were 11 .1% .The positive rate of ampC of Klebsiella pneumoniae were 23 .7% ,mainly DHA type ,counting for 78 .3% ,CIT type count‐ing for 21 .7% .The results of DNA sequencing showed that there were 18 strains DHA‐1 type and 1 strain ampC gene type of Morganella morganii in DHA type strains ,the concordance rate was 97 .0% ,10 CIT type strains was CMY‐2 type ,1 strain was CMY‐42 ,one strain was CMY‐4 type ,EBC type was ampC gene type of Enterobacter cloacae ,the concordance rate was 99 .0% .A total of 32 strains of gene sequencing were registered as KJ127248 - KJ127279 in GenBank .Conclusion The main genotypes of plasmid‐mediated ampC enzyme produced by Escherichia coli and Klebsiella pneumoniae were CMY‐2 and DHA‐1 respectively .

Molecular diagnosis of β-thalassemia / 中华检验医学杂志

β-thalassemia is one of the most common monogenic genetic disorders in the world,and represents a major public health problem among humans.This review descrihes the latest technical development of molecular diagnosis of β-thalassemia from the perspective of clinical settings.Related techniques including reverse dot blot,minisequencing,real-time PCR,and non-invasive prenatal diagnosis,etc.are discussed regarding their principles,features and trends.

Application and evaluation of a probe melting analysis-based assay for detection of Isoniazid-resistant mutations in Mycobacterium tuberculosis / 中华检验医学杂志

Objective To validate the performance of a PMA-based assay for detection of INH-resistant mutations in MTB and investigate the mutation characteristic of INH-resistance.Methods The MTB standard strain H37Rv was from National Tuberculosis Reference Laboratory,1 wild-type strain and 1 katG S315T ACC mutant strain were from Xiamen CDC,7 MTB INH-resistant strains with known INH resistance mutations were from Shenzhen Center for Chronic Disease Control,Henan CDC,No.309 Hospital of PIA and Xiamen CDC.707 MTB clinical isolates were from Xiamen CDC,Xiamen No.1 Hospital and Zhangzhou CDC,126 sputum samples were from Xiamen Tongan CDC.The genomic DNA of the MTB standard strain H37Rv,7 MTB INH-resistant strains and 833 clinical samples,were extracted with Xiamen Zeesan Mycobacterium tuberculosis Isoniazid-resistance Mutation Test Kit using the thermal lysis method.The genomic DNA of 1 wild-type strain and 1 katG S315T ACC mutant strain were extracted with AxyPrep Bacterial Genomic DNA Miniprep Kit.The mutations were discriminated by the △Tm between the samples and wild type control in katG315 codon,inhA promoter -17 to -8 region,ahpC promoter region -44 to -30 and - 15 to 3,and inhA94 codon.A 10-fold dilution series of MTB DNA from 3 × 105 to 300 copies/ reaction obtained from a wild-type strain and a katG S315T ACC mutant strain,respectively,were prepared to determine the analytical sensitivity.Seven MTB INH-resistant strains with 9 predetermined mutations were used for the analytical specificity assay,and 5 mutants of which were used for the repeatability assay.The clinical detection performance of PMA assay were confirmed by the sequencing method in 833 samples.Results Results could be obtained within 3 hours from DNA extraction to PMA assay,including 46 samples in a standard 96-well real-time PCR instrument simultaneously.The analytical sensitivities of PMA were 300 copies/reaction for both the wild-type strain and katG S315T ACC mutant strain.Nine INH-resistant point mutations could be discriminated and 5 of which had standard deviations of melting temperature less than 0.5 ℃.Fully concordant results of mutant locus between PMA assay and sequencing were obtained in all 162 mutant samples.INH-resistant mutations in the four loci were found in 19.4％ (162/833) samples by PMA assay in Xiamen and Zhangzhou.Among the 14 lNH-resistant mutant types detected,katG S315T ( AGC→ACC),inhA promoter - 15C→T and katG S315N (AGC→AAC) accounted for 83.3％ (135/162) of the overall mutations.

Probe melting analysis for rapid detection of ethambutol-resistant mutations in Mycobacterium tuberculosis clinical isolates / 中华检验医学杂志

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.