The Research Of Immunologic And Molecular Biological Detection Method For Cronobacter Spp.

Cronobacter spp. are foodbome opportunistic pathogens of the family Enterobacteriaceae. They can cause necrotizing enterocolitis, sepsis, and meningitis in all age groups, especially infants with low weight or within month. The fatality rate of the infection is up to 80%. This opportunistic pathogen had been epidemiologically linked to contaminated powdered infant formula (PIF). In 2004, Cronobacter spp. had been identified as pathogens of A group in PIF (only two pathogens included in this group and another one was Salmonella spp.) by FAO/WHO. Conventional culture-based method played an important role in the detection of Cronobacter spp. in China. It usually consisted of a series of steps, including non-selective enrichment, selective enrichment,differential plating, and final biochemical confirmation. However, at least 5 days were needed and the procedure was complex. Furthermore, the sensitivity of it was relatively low. Culture-based methods mainly focus on the detection of Cronobacter genus.However, the pathogenicity and virulence varied with different specie in Cronobacter genus. C. sakazakii ST4 caused the majority of serious neonatal and infant infections.Therefore, it is important to develop rapid, sensitive and specific detection methods for Cronobacter spp. and C. sakazakii.Our research of rapid, sensitive and specific detection methods for Cronobacter spp.could be divided into several parts:(1) Construction and expression of a single-chain variable fragment scFvH81 specific to Cronobacter spp. from hybridoma.Total RNA of hybridoma was extracted and reverse transcribed. VH gene candidates and nine VL gene candidates with correct size amplified from the cDNA were sequenced and analyzed. Among them, VH81and VL11 showed intact open reading frames and a better homology. Then a single chain FV named scFvH81 was constructed. The scFvH81 was prepared by denaturation and renaturation of inclusion body. The process was fast(only 5 days was needed ), simple and low-cost. ScFvH81 was characterized, the result of specificity test showed that scFvH81 could be used in the detection and the value of Kaff was 2.39±0.06 × 106 M-1.(2) The analysis of scFvH81 by bioinformatics and construction of dsFv.The sequence of scFvH81 was analyzed by the IMGT/V-QUEST, the V (D) J rearrangement mechanism of it and the embryonic genes were determined. The predicted isoelectric point of scFvH81 was 7.05 and the pH of renaturation buffer was adjusted to 8 to provide a better environment for renaturation. The secondary structure and 3-D model were predicted by SOPMA and I-TASSAR,respectively. The interface of antigen-antibody compound was analyzed and the key amino acids were determined.They were usually at the beta-turn of scFvH81. A disulfide bond was then induced into the scFvH81. The sites of mutation in 3-D model was near in space and avoided key amino acids. However, the spatial structure of dsFv had changed and showed a bad specificity compared with scFvH81. Therefore more factors should be considered in the construction of dsFv.(3) Mining for specific primers for PCR detection of Cronobacter spp. by a bioinformatic approachThe genome sequence of C. sakazakii BAA-894 in the GenBank database was compared with other organisms using BLAST. Twenty-two DNA fragments unique to Cronobacter spp. were identified, and primers targeting these sequences were designed.Primers set N2 was found to be specific to Cronobacter genus and a PCR detection method with good ability of interference was then developed. The detection limit of it was 102 CFU/mL for pure cell culture and 128 fg/p.L for genome DNA. C. sakazakii was detected in artificially contaminated PIF with initial cell density of 3.5 × 100 after 8 h.(4) Mining for species-specific primers for PCR detection of C. sakazakii by a bioinformatic approachThe genome sequence of C. sakazakii BAA-894 in the GenBank database was compared with that of other organisms using BLAST. Thirty-eight DNA fragments unique to C. sakazakii were identified, and primers targeting these sequences were designed. Finally, three primer sets (CS14, CS21, and CS38) were found to be specific for C. sakazakii by PCR verification. The detection limit of PCR assays using the three pairs of primers were 1.35 pg/?L, 135 fg/?L, 135 fg/?L for genomic DNA, and 5.5 × 105 CFU/mL, 5.5 × 103 CFU/mL, 5.5 × 103 CFU/mL for pure cell culture, respectively, as compared to 13.5 pg/?L and 5.5 × 105 CFU/mL for primer set SpeCronsaka, which has been previously described. C. sakazakii was detected in artificially contaminated powdered infant formula (PIF) by PCR using both primer sets CS21 and CS38 after 8 h of enrichment. The detection limit was 5.5 × 10-1 CFU /10g PIF.(5) Development of a real-time PCR method with internal amplification control for rapid detection of C. SakazakiiThe primers set CS38 was chosen to develop a real-time PCR method for rapid detection of C. Sakazakii. In this method, an internal amplification control (IAC) was constructed and added in the reaction system. The detection limit of PCR assays using the three pairs of primers was 33 fg/?L for genomic DNA, and 4.7×101CFU/mL using pure cultures of the bacteria. C. sakazakii was detected in artificially contaminated PIF with initial cell density of 3.5 × 1000CFU/mL after 6 h.