Performance of probe polymerization-conjunction-agarose gel electrophoresis in the rapid detection of KRAS gene mutation.

This study aimed to develop a simple and rapid method to detect KRAS gene mutations for conventional clinical applications under laboratory conditions. The genotype of mutation sites was determined based on the occurrence of target bands in the corresponding lanes of the reaction tubes through polymerization-conjunction of the probes, probe purification and amplification, and agarose gel electrophoresis. Circulating DNA samples were obtained from the plasma of 72 patients with lung cancer, which were identified based on six mutation sites (G12S, G12R, G12C, G12D, G12A, and G12V) of codon 12 of the KRAS gene. The detection results were compared with direct sequencing data. The proposed detection method is characterized by simple operation, high specificity, and high sensitivity (2%). This method can detect the mutations of three samples at G12S, G12R, and G12A. In the direct sequencing spectra of these samples, the genotype could not be determined due to the lack of evident sequencing peaks that correspond to the basic group of mutations. In conclusion, a simple and rapid method was established based on probe polymerization-conjunction-agarose gel electrophoresis for detecting KRAS gene mutations. This method can be applied to the conventional mutation detection of inhomogeneous samples.

Molecular epidemiology and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus isolates in Xiangyang, China.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide. This study aimed to investigate the antimicrobial susceptibility and molecular epidemiological characteristics of MRSA strains in Xiangyang, China, during 2012-2014. METHODS: Eighty non-duplicate S. aureus isolates from clinical specimens were collected from four tertiary hospitals. MRSA strains were identified and were tested for antibacterial susceptibility. Staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal protein A (spa) typing were performed to explore molecular characteristics. RESULTS: Among the 80 S. aureus isolates, 43 MRSA (53.8%) were detected. MRSA strains exhibited resistance against non-ß-lactam antibiotics to varying degrees. SCCmec type III was the predominant type (39/43; 90.7%), and the remainder were SCCmec type IVa (4/43; 9.3%). Thirteen MLST types were found, mainly ST239 (12/43; 27.9%) and ST59 (7/43; 16.3%). Fifteen spa types were found, mainly t437 (13/43; 30.2%) and t030 (6/43; 14.0%). PFGE grouped the 43 MRSA isolates into five types. SCCmecIII-ST239-t030/t632 and SCCmecIII-ST59/ST338-t437 were the dominant epidemic clones in this region. ST239-t030/t632/t037 was the epidemic clone with the most serious drug resistance. CONCLUSIONS: This region presented a high MRSA rate and the MRSA isolates demonstrated strong antimicrobial resistance. The existence of four strains of community-acquired MRSA (SCCmec type IVa) indicated the dissemination of MRSA strains from the community to hospitals. The epidemic situation and drug resistance of MRSA should be regularly monitored. Effective measures should be adopted to prevent and control the occurrence of infection in hospitals.

Detection of mutations by fill-in ligation reaction with enzyme-linked immunosorbent assay for rapid medical diagnosis.

Several approaches for parallel genotyping have been developed with increasingly available information on DNA variation. However, these methods require either complex laboratory procedures or expensive instrumentation. None of these procedures is readily performed in local clinical laboratories. In this study, we developed a flexible genotyping method involving fill-in ligation reaction with enzyme-linked immunosorbent assay successfully applied to detect important single-nucleotide polymorphisms (SNPs) for EGFR c.2573T > G (L858R), EGFR c.2582T > A (L861Q), and EGFR c.2155G > T (G719C). This assay exhibited excellent specificity, with a sensitivity as low as 0.5%. Eight out of 62 clinical samples were identified as heterozygotes for the SNP site of L858R, whereas only two samples were identified as heterozygotes by direct sequencing. The developed method enabled accurate identification of SNP in a simple and cost-effective manner adapted to routine analysis.