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Development and validation of a prokaryotically expressed foot-and-mouth disease virus non-structural protein 2C'3AB-based immunochromatographic strip to differentiate between infected and vaccinated animals.

Wu L, Jiang T, Lu ZJ, Yang YM, Sun P, Liang Z, Li D, Fu YF, Cao YM, Liu XT, Liu ZX - Virol. J. (2011)

Bottom Line: The coincidence rate of cattle negative serum, cattle vaccinated serum, cattle infected serum was 100%, 96.7%, 98.0%, respectively.The coincidence rate of sheep negative serum, sheep infected serum was 97.6%, 96.3%, respectively.The strip was shown to be of high specificity and sensitivity, good repeatability and stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Veterinary Etiologic Biology, National Foot-and-Mouth Disease Reference Laboratory of China, Key laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, Gansu, China.

ABSTRACT

Background: Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cattle, pigs, sheep, goats, and many cloven-hoofed wild animals. FMDV serotypes O and Asia 1 have circulated separately in China during the last fifty years, and eliminating infected animals and vaccination are the main policies to prevent and control FMD. Antibodies to NSPs exist in infected animals, and were utilized to differentiate between infected and vaccinated animals. The reliability of detection of 3AB or 3ABC antibodies is higher than that of other NSPs. The test of 3AB is still credible because 3C protein's immunogenicity is the weakest. The 2C protein, immediately N-terminal of 3AB, was used to differentiate between infected and vaccinated animals. The use of the immunochromatographic strip is facile for clinical laboratories lacking specialized equipment and for rapid field diagnosis.

Results: In this study, an immunochromatographic strip with non-structural protein (NSP) 2C'3AB was developed and validated to differentiate foot-and-mouth disease infected from vaccinated animals. A part of N-terminal of 2C protein gene and whole 3AB gene were connected and prokaryotically expressed as the antigens labeled with colloidal gold was used as the detector, the 2C'3AB protein and rabbits anti-2C'3AB antibodies were blotted on the nitrocellulose(NC) membrane for the test and control lines, respectively. 387 serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial 3ABC antibody ELISA kit. The coincidence rate of pigs negative serum, pigs vaccinated serum, pigs infected serum was 100%, 97.2%, 95.0%, respectively. The coincidence rate of cattle negative serum, cattle vaccinated serum, cattle infected serum was 100%, 96.7%, 98.0%, respectively. The coincidence rate of sheep negative serum, sheep infected serum was 97.6%, 96.3%, respectively. The strip was shown to be of high specificity and sensitivity, good repeatability and stability.

Conclusion: These data suggest that the immunochromatographic strip is a useful tool for rapid on-site diagnosing animals infected foot-and-mouth disease virus.

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Related in: MedlinePlus

The schematic construction of recombinant expression plasmid pET-30a-2C'3AB.
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Figure 3: The schematic construction of recombinant expression plasmid pET-30a-2C'3AB.

Mentions: Two couples of primers(see Figure 1) were designed with DNAstar and Oliogo6.0 softwares based on the sequence of FMDV O/China/99(Genbank No.AF506822) to amplify 2C'3AB gene using standard protocols. The 2C'3AB PCR product was ligated into pGEM-T (pGEM-2C'3AB; see Figure 2). pGEM-2C'3AB was restricted with NcoI and SalI, the insert gel purified and ligated into pET-30a, similarly restricted, to form pET-30a-2C'3AB (Figure 3). E. coli strain BL21(DE3)pLysS was transformed with pET-30a-2C'3AB. Isopropylthiogalactoside (IPTG) was added to induce expression of the 2C'3AB protein. The soluble 2C'3AB polypeptide in the lysate was purified by a HisTrap HP affinity chromatography column. SDS-PAGE and Western blotting were used to detect the expression product[1]. The concentration of the purified 2C'3AB protein was measured with BCA (Bicinchoninic Acid) protein quantitative kit (Beyotime, Shanghai)


Development and validation of a prokaryotically expressed foot-and-mouth disease virus non-structural protein 2C'3AB-based immunochromatographic strip to differentiate between infected and vaccinated animals.

Wu L, Jiang T, Lu ZJ, Yang YM, Sun P, Liang Z, Li D, Fu YF, Cao YM, Liu XT, Liu ZX - Virol. J. (2011)

The schematic construction of recombinant expression plasmid pET-30a-2C'3AB.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094302&req=5

Figure 3: The schematic construction of recombinant expression plasmid pET-30a-2C'3AB.
Mentions: Two couples of primers(see Figure 1) were designed with DNAstar and Oliogo6.0 softwares based on the sequence of FMDV O/China/99(Genbank No.AF506822) to amplify 2C'3AB gene using standard protocols. The 2C'3AB PCR product was ligated into pGEM-T (pGEM-2C'3AB; see Figure 2). pGEM-2C'3AB was restricted with NcoI and SalI, the insert gel purified and ligated into pET-30a, similarly restricted, to form pET-30a-2C'3AB (Figure 3). E. coli strain BL21(DE3)pLysS was transformed with pET-30a-2C'3AB. Isopropylthiogalactoside (IPTG) was added to induce expression of the 2C'3AB protein. The soluble 2C'3AB polypeptide in the lysate was purified by a HisTrap HP affinity chromatography column. SDS-PAGE and Western blotting were used to detect the expression product[1]. The concentration of the purified 2C'3AB protein was measured with BCA (Bicinchoninic Acid) protein quantitative kit (Beyotime, Shanghai)

Bottom Line: The coincidence rate of cattle negative serum, cattle vaccinated serum, cattle infected serum was 100%, 96.7%, 98.0%, respectively.The coincidence rate of sheep negative serum, sheep infected serum was 97.6%, 96.3%, respectively.The strip was shown to be of high specificity and sensitivity, good repeatability and stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Veterinary Etiologic Biology, National Foot-and-Mouth Disease Reference Laboratory of China, Key laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, Gansu, China.

ABSTRACT

Background: Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cattle, pigs, sheep, goats, and many cloven-hoofed wild animals. FMDV serotypes O and Asia 1 have circulated separately in China during the last fifty years, and eliminating infected animals and vaccination are the main policies to prevent and control FMD. Antibodies to NSPs exist in infected animals, and were utilized to differentiate between infected and vaccinated animals. The reliability of detection of 3AB or 3ABC antibodies is higher than that of other NSPs. The test of 3AB is still credible because 3C protein's immunogenicity is the weakest. The 2C protein, immediately N-terminal of 3AB, was used to differentiate between infected and vaccinated animals. The use of the immunochromatographic strip is facile for clinical laboratories lacking specialized equipment and for rapid field diagnosis.

Results: In this study, an immunochromatographic strip with non-structural protein (NSP) 2C'3AB was developed and validated to differentiate foot-and-mouth disease infected from vaccinated animals. A part of N-terminal of 2C protein gene and whole 3AB gene were connected and prokaryotically expressed as the antigens labeled with colloidal gold was used as the detector, the 2C'3AB protein and rabbits anti-2C'3AB antibodies were blotted on the nitrocellulose(NC) membrane for the test and control lines, respectively. 387 serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial 3ABC antibody ELISA kit. The coincidence rate of pigs negative serum, pigs vaccinated serum, pigs infected serum was 100%, 97.2%, 95.0%, respectively. The coincidence rate of cattle negative serum, cattle vaccinated serum, cattle infected serum was 100%, 96.7%, 98.0%, respectively. The coincidence rate of sheep negative serum, sheep infected serum was 97.6%, 96.3%, respectively. The strip was shown to be of high specificity and sensitivity, good repeatability and stability.

Conclusion: These data suggest that the immunochromatographic strip is a useful tool for rapid on-site diagnosing animals infected foot-and-mouth disease virus.

Show MeSH
Related in: MedlinePlus