Guozhen Lin

Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp.

A novel loop-mediated isothermal amplification (LAMP) assay was established to detect Brucella species DNA in milk and blood samples of animals and humans. This LAMP assay based on the sequence of highly repetitive omp25 gene was able to detect 9fg/μl Brucella spp. DNA with high sensitivity, which was 10 times higher than the nested PCR. The LAMP was evaluated for its specificity using 19 strains of six Brucella species and 28 related non-Brucella micro-organism strains as controls. The target 19 Brucella strains were all amplified, and no cross-reaction was found with all the non-Brucella micro-organism strains. Both nested PCR and LAMP assays were then used to detect Brucella spp. DNA in 78 milk samples and 113 blood samples from animals and 11 blood samples from humans, and the established LAMP assay yielded 99.0% concordance rate with the nested PCR. The LAMP assay should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of Brucellosis.

Serological detection of bovine ephemeral fever virus using an indirect ELISA based on antigenic site G1 expressed in Pichia pastoris.

An indirect ELISA for the serological detection of bovine ephemeral fever virus (BEFV) infection in cattle is described in which a glycosylated protein of approximately 25 kDa (including the G1 antigenic site of the virus glycoprotein) expressed in Pichia pastoris GS115 was used as the coating antigen. The optimal concentration of coated antigen was 0.3 microg/well at a serum dilution of 1:40. The optimal positive threshold value of the assay was 1.88, as derived from receiver operating characteristic curve analysis. The test had 100% sensitivity and 96.7% specificity when compared with a micro-neutralisation test using 336 positive and 180 negative sera to BEFV, respectively. The inter-assay and intra-assay coefficients of variation for 15 sera were both <5.8% and there was no evidence of cross-reactivity between the tested coating antigen and antibodies to related rabies virus. The ELISA is an inexpensive and rapid serological detection method that would be suitable for screening for BEFV infection on a large scale.

Development and application of G1-ELISA for detection of antibodies against bovine ephemeral fever virus.

The gene encoding antigenic site G(1) of bovine ephemeral fever virus (BEFV) was highly expressed in the host cell Escherichia coli. An indirect G(1)-ELISA with the recombinant protein as the coating antigen was established to detect antibodies against BEFV. The result revealed that the optimal concentration of the coated antigen was 0.5 microg/well and the dilution of serum was 1:20. It was optimal that sera with P/N >or= 2.2 were considered positive, P/N <or=2.0 negative, and between 2.0 and 2.2 ambiguous. The G(1)-ELISA method gave a sensitivity of 97.6% and a specificity of 98.6% by testing 590 field serum samples. These results suggest that the G(1)-ELISA may be a good alternative tool for seroepidemiological surveys.

A reverse-transcription, loop-mediated isothermal amplification assay for detection of bovine ephemeral fever virus in the blood of infected cattle.

A novel reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) assay for the detection of bovine ephemeral fever virus (BEFV) was developed and evaluated in this study. The RT-LAMP assay exhibited higher sensitivity when compared with conventional reverse-transcription polymerase chain reaction (RT-PCR) and virus isolation methods. The specificity of the assay was determined by digestion of the RT-LAMP products with restriction enzyme and detection of BEFV serogroup rabies virus (RV). Using RT-LAMP, RT-PCR and virus isolation methods, 36 blood samples were tested and the results indicated that RT-LAMP could detect early infection with BEFV. The RT-LAMP method is useful for the diagnosis of BEFV infection in blood samples.

Loop-mediated isothermal amplification assay targeting the ompA gene for rapid detection of Riemerella anatipestifer

A novel loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of Riemerella anatipestifer (RA) infection. The LAMP assay exhibited a higher sensitivity than conventional polymerase chain reaction (PCR) and microbial isolation. The specificity of the assay was determined by restriction enzyme digestion of the LAMP products and detection of Escherichia coli, Salmonella enterica and Pasteurella multocida. The LAMP assay was able to detect RA effectively in samples of the reference strains, isolated strains and infected duck brains. This assay is a useful tool for the diagnosis of RA infection in the clinical setting.

Discovery and characterization of gene cassettes-containing integrons in clinical strains of Riemerella anatipestifer

Forty-eight prevalent strains of Riemerella anatipestifer (RA) isolated in China were tested for susceptibility to eighteen antibiotics and investigated for the frequencies and characteristics of integrons and gene cassettes. All isolates were resistant to between three and ten antimicrobial drugs. Forty-seven isolates contained class 1 integron (97.92%), and 15 of the 47 isolates contained class 2 integron (31.25%). Class 3 integron was not detected in the strains analysed. Three different cassette arrays (aadA1, aadA5 and aacA4-aadA1) of class 1 integron and one gene cassette (sat2-aadA1) of class 2 integron were discovered. Three out of the four cassette arrays were novel, with the exception of aadA5. The location of integrons was confirmed by transforming extracted plasmids into an integron-negative strain of Escherichia coli (E. coli) BL21 (DE3). Class 1 integrons were always discovered in plasmids, while class 2 integrons could be located on plasmids or in the chromosome. This is the first description of class 2 integrons, three novel cassette arrays and the location of integrons in RA species.

An indirect ELISA of classical swine fever virus based on quadruple antigenic epitope peptide expressed in E.coli

In this study, a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV) E2 glycoprotein was expressed in E. coli to a obtain target protein. This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs. The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve (ROC) analysis based on 30 negative sera and 80 positive samples. The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination (IHA) test. The inter-assay and intra-assay coefficients of variation (CVs) for 16 sera were both ⩽6.8%. No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus (BVDV) antibodies was observed.

Immunization trials with an avian chlamydial MOMP gene recombinant adenovirus

Avian chlamydiosis (AC) is an infectious disease caused by Chlamydophila psittaci. Named after psittacosis or ornithosis, the bacterium mainly harms domestic and wild birds. Early in the 20th century, it was fashionable to keep parrots as domestic pets. However, many of these birds were affected with ornithosis, leading to sporadic human outbreaks of AC. Hundreds of people died of AC in America, Europe and Africa 1, 2. AC exists in many parts of the world and can infect almost all bird species, including domestic poultry 3. When affected with AC, the mortality rate in infant birds is high, but decreases as birds reach adulthood. Between the 1960’s to 1970’s, the poultry industry in Europe suffered serious losses due to AC 4,5. Not restricted to Europe, AC cases involving duck, dove, chicken, turkey, goose and wild birds have also been reported in China. Indeed a mean positive rate of 10.96% AC has been reported in 18 provinces1. Furthermore, the incidence of AC cases appears to be increasing in some provinces. However, , despite this, no effective vaccine has yet been developed to control the disease. In the current study, specific pathogen free (SPF) chicks were immunized with an avian chlamydial major outer membrane protein (MOMP) gene recombinant adenovirus vector vaccine developed in our laboratory6. The minimal infection dose of the virulent strain, the most effective inoculation route of the vaccine and the minimal effective immunization dose were determined. Vaccine potency, security, protective period, and the conservation period of the vaccine were also examined.

Expression and antigenic characterization of the epitope-G1 of the Bovine ephemeral fever virus glycoprotein in Pichia pastoris

The epitope-G1 gene of Bovine ephemeral fever virus (BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1. Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS115. The recombinant P. pastoris strains were selected by a G418 transformation screen and confirmed by PCR. After being induced with methanol, an expressed protein with 26 kDa molecular weight was obtained, which was much bigger than the predicted size (15.54 kDa). Deglycosylation analysis indicated the recombinant G1 was glycosylated. Western blot and ELISA tests, as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity. The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.

Secretory Expression of E2 Main Antigen Domain of CSFV C Strain and the Establishment of Indirect ELISA Assay

The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21-CodonPlus (DE3)-RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM4B by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was ODtested serum / ODnegative serum ⩾ 2.1. The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.