To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

OBJECTIVETo construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics.

METHODSThe gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining.

RESULTS AND CONCLUSIONWe established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

DNA Primers ; Escherichia coli O157 ; genetics ; Escherichia coli Proteins ; genetics ; Gene Deletion ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction

Objective To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. Methods The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157:H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD60 value and Giemsa staining. Results and Conclusion We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

Objective To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. Methods The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157:H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD60 value and Giemsa staining. Results and Conclusion We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

Objective To explore the complications and the treatments of repeated cesarean delivery in scar uterus pregnancy accompanied by the placenta praevia. Methods We performed a retrospective study in 6832 cases underwent first cesarean delivery,within which 201 cases were accompanied by the placenta praevia;and 337 cases underwent repeated cesarean deliver, within which 26 cases were accompanied by the placenta praevia. All subjects accepted cesarean delivery from January2006 toApril 2010 in our hospital. Results The occurrence rate of placenta previa was significantly higher in the repeated cesarean delivery than first-ever cesarean delivery (7. 72% vs 2. 94%, x2 = 22. 33, P ＜ 0. 01 ) , especially the occurrence of complete placenta previa (42. 30% vs 0. 00%, x2 = 80. 43, P ＜ 0. 01 ). The rates of uterus rupture, placenta accreta, postpartum hemorrhage and hysterectomy (r = 26. 92% ,23.08% ,26. 92% and 7. 69%, respectively) in repeated cesarean delivery in scar uterus pregnancy accompanied by the placenta praevia were significantly higher than those of the cases ( r = 2. 57% ,0. 32%, 1.29% and 0. 00%, respectively ) had repeated cesarean delivery in scar uterus pregnancy but no placenta previa ( x2 = 27.97,50. 41,42. 16,12. 79, respectively, Ps ＜ 0. 01 ). Conclusion The incidence of placenta previa increased in scar uterus pregnancy, especially the complete placental previa.Scar uterus pregnancy accompanied by the placenta praevia are more likely to occur uterus rupture,placenta accreta,postpartum hemorrhage and had hysterectomy. Obstetricians should give more effort to reduce the cesarean section rate,improve the quality of medical care.