Optimized MOL-PCR for Characterization of Microbial Pathogens.

Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium.

Identification of Salmonella H antigens by xTAG technology / 中华微生物学和免疫学杂志

Objective To develop a microsphere-based suspension array for simultaneous detec-tion and identification of Salmonella H antigens by using Luminex xTAG technology and to evaluate its capa-bility in serotyping Salmonella strains. Methods The fliC and fljB genes, encoding the H antigen of Salmo-nella, were selected as the target genes. Universal upstream primers were designed based on the highly con-served regions of fliC and fljB genes, and the corresponding specific reverse primers were designed based on the variable regions. While synthesizing, the 5′end of each upstream primer was labeled with biotin and the 5′end of each specific reverse primer was modified with its certain TAG sequence. After amplified and la-beled with biotin and TAG sequence, the PCR products of specimens were hybridized with the mixture of va-rious MagPlex-xTAGTM microspheres. Each set of microspheres contained its unique anti-TAG sequences. The results of hybridization were analyzed by using Luminex MagPix reader system and the median fluores-cence intensity ( MFI) was reported. The H antigens of 145 Salmonella strains were identified with this de-veloped xTAG suspension array, and the results were compared with those obtained by using traditional ser-um agglutination test. Results The PCR products of different H antigens ranged from 94 bp to 245 bp and could be identified by hybridizing with MagPlex-xTAGTM microspheres. There was no cross-reaction between different H antigens or with DNAs derived from Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus and Shigella flexneri. Compared with the traditional serum agglutination test, the sensitivity and specificity of the xTAG suspension array in the identification of H antigens of 145 Salmonella strains were 95. 1% and 100%, respectively. Conclusion The developed xTAG suspension array was a specific, accurate and effective method for simultaneous detection and identification of 31 H antigens of common Salmonella serovars strains. It could be used for determining the H antigens of more than 90 Salmonella strains within 5 hours.

Epidermal growth factor receptor mutations in Chinese patients with laryngeal squamous cell carcinoma.

CONCLUSION: The epidermal growth factor receptor (EGFR) mutation is rare in patients with laryngeal squamous cell carcinoma (LSCC) in China. OBJECTIVE: To determine the incidence of EGFR mutations in patients with LSCC who underwent surgical resection in mainland China. METHODS: xTAG technology was used to detect the EGFR exon 19, exon 20, and exon 21 mutations in 132 patients with LSCC who underwent surgical treatment in our hospital from 2010 to 2013. RESULTS: Of the 132 LSCC specimens examined, only 1 specimen was found to be positive for EGFR exon 20 mutation (0.76%). The mutation was p.T790M in exon 20. Two LSCC specimens were positive for EGFR exon 21 mutation (1.52%). The mutation was p.L858R in exon 21. None of the samples was found to be positive for EGFR exon 19 mutation.