Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.
Abstract. A latex agglutination test (LAT) for detecting antibody against Bluetongue virus (BTV) in ruminants was developed using latex beads coupled with recombinant VP7 protein. Compared with competitive enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the LAT were 99.0% and 93.0%, respectively. There was excellent agreement between the results obtained by competitive ELISA and the LAT (kappa 5 0.930). Because it is rapid and easy to use, the LAT could be used for BTV antibody detection, especially for screening many serum samples.Key words: Bluetongue virus; latex agglutination; recombinant VP7 antigen.Bluetongue virus (BTV), the prototype virus of the genus Orbivirus in the family Reoviridae, 12 is an arthropod-borne virus that infects both domestic and wild ruminants, causing a noncontagious infectious disease or bluetongue (BLU), which has been reported in every continent except Antarctica.5,14 At present, 24 serotypes of BTV have been characterized. 12All ruminant species can be infected with BTV, but improved breeds of sheep and some species of deer tend to develop particularly severe clinical signs of disease with mortality about 50%-70% in infected sheep flocks. 6,8 In addition to morbidity and mortality, BLU disrupts the international trade in animals and animal products because countries free of BTV infection frequently restrict the importation of ruminants and their genetic products from BTV-endemic areas. 12Bluetongue virus is a double-stranded RNA (dsRNA) virus and its genome consists of 10 dsRNA segments that encode 4 nonstructural proteins (NS1-NS3, and NS3a) and 7 structural proteins (VP1-VP7).4 Among the 7 structural proteins, VP7 has been identified as the group-specific antigen of BTVs by different immunoassays.2,13 The VP7 protein was the antigen used for developing group-specific immunoassays, such as competitive enzyme-linked immunosorbent assay (ELISA), which is more specific and sensitive than the agar gel immunodiffusion (AGID) test based on whole virus antigen. 1,7,10,11 In the current study, a latex agglutination test (LAT), based on latex beads coupled with recombinant VP7 protein, was established for BTV antibody detection in serum samples. The LAT yielded higher sensitivity than that of AGID based on whole BTV antigen, and the specificity and sensitivity of the LAT were comparable with that of a commercial competitive ELISA. Because it is rapid and easy to use, the LAT is suitable for detecting anti-BTV antibody at the farm.The VP7 gene fragment, amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of BTV serotype 1, was cloned into pBAD/Thio plasmid a to form the recombinant expression vector pBAD/Thio BTV VP7. The VP7 protein was expressed in Escherichia coli LMG194 cells induced by L-arabinose at a final concentration of 200 mg/ml.9 The expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay. Coomassie blue staining showed that VP7 fusion protein was expres...
This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻⁵(= 280ELD₅₀) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻⁴(=2800 ELD₅₀). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.
A new competitive bead immunoassay (CBIA) based on Luminex technology for detecting clenbuterol in urine was reported. The carboxylated fluorescent beads were directly coated with clenbuterol derivatives without carrier protein spacer. Clenbuterol antibody was biotinylated, which was used for clenbuterol detection in combination with the functionalized bead and streptavidin-phycoerythrin (SAPE). The effects of spacer on the CBIA method were investigated. The results indicated that the presence of small molecular spacer between bead and hapten improved the assay sensitivity and the hydrophilic spacer (glycine) was better than the hydrophobic spacer (m-aminobenzoic acid) for this CBIA method. The study affirms the importance of the judicious choice of hapten derivatives in the CBIA method for detecting small molecule drug based on Luminex technology. The method could be used for clenbuterol detection in livestock urine and possible for the simultaneous detection of multiple veterinary drugs.
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