Objective　To shorten the detection time of Staphylococcus aureus (SA), this study established a rapid detection method for the thermostable nuclease nuc gene and mecA resistance gene of SA based on recombinase aided amplification (RAA) combined with lateral flow strips (LFS). Methods Efficient RAA primers and probes were designed and screened based on the conserved sequences of the nuc gene in the SA genome and the mecA gene on the staphylococcal SCCmec removable genetic element, and then, the reaction temperature and for the simultaneous detection of nuc and mecA genes by time of RAA-LFS were verified. Sensitivity, specificity, and comparison with quantitative real-time PCR (qPCR) were also assessed. A prospective evaluation was conducted using 52 vials of non-repeat positive blood cultures from two tertiary hospitals. Results The RAA-LFS was able to amplify both nuc and mecA genes under the reaction conditions of 20-45 °C for 15-30 minutes, with a detection limit of 10² CFU/mL. A total of 50 clinically isolated non-SA strains were validated with 100% specificity for both nuc and mecA genes. Of the 30 methicillin-resistant Staphylococcus aureus (MRSA) and 30 methicillin-susceptible Staphylococcus aureus (MSSA) strains preserved in our laboratory, all were positive for the nuc gene, and 30 strains were positive for the mecA gene. The positive and negative concordance rates with qPCR were both 100%, with a consistency test Kappa value of 1. In a prospective analysis of the 52 vials of positive blood cultures, the identification and antibiotic susceptibility test results of 16 strains of MSSA and 6 strains of MRSA showed a 100% concordance rate with the results obtained using the Mérieux VITEK-2 compact microbiological detection system. Conclusions We combined nucleic acid release agents, RAA, and lateral flow strips to develop a simple, rapid, and highly sensitive assay applicable for SA and mecA resistance gene detection in colonies or positive blood culture bottles.

In June 2016,a disease among the cultured rock carp (Procypris rabaudi) in Yongchuan of Chongqing Municipality occurred.The aim of this study was to investigate biological characteristics and provide reference for Aeromonas veronii identification diagnosis and treatment.Pathogenic bacteria strain YY01 from the dying fishes were examined and isolated.Strain YY01's taxonomic status was identified by observing the morphology,studying the physiological and biochemical characters and sequencing the 16S rRNA and housekeeping gene gyrB.Its pathogenicity was checked by artificial infection experiment and virulence genes.Furthermore,effective medicine was detected by drug sensitivity.The 16S rRNA and gyrB gene sequence of the strain YY01 was more than 99％ homology with that of Aeromonas veronii,suggesting that the pathogen was Aeromonas veronii,which was also identified by the results of biochemical analysis.The LD50 of strain YY01 to rcok carp was 5.06 × 104 CFU/g.Four virulence genes were detected from YY01,including aerolysin (aer),hemolysin (Hly),Outer Membrane Protein Gene A (OmpA) and adhesion (Aha) genes.Antibiotic sensitivity assays showed that among 40 antibiotics tested,22 were sensitive and 11 were resistant.In conclusion,the strain YY01 is identified as Aeromonas veronii and it is proved to have strong pathogenicity.

Objective:To construct GJB2 gene mutaitons common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutaitons in GJB2 gene. Method: Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutaiton methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutaions were inserted into pEG-FP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were trans-fected with the recombinant DNA samples by the liposome complex method. Results The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence singals were distributed uniformly in cytoplasm. Conclusion; GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.

OBJECTIVE: To construct GJB2 gene mutations common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutations in GJB2 gene. METHOD: Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutation methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutations were inserted into pEGFP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were transfected with the recombinant DNA samples by the liposome complex method. RESULT: The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence signals were distributed uniformly in cytoplasm. CONCLUSION GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.
Asian Continental Ancestry Group ; genetics ; Connexin 26 ; Connexins ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Sequence Deletion