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Influenza Other Respir Viruses. 2016 Mar;10(2):141-9. doi: 10.1111/irv.12370.

Simultaneous detection of four different neuraminidase types of avian influenza A H5 viruses by multiplex reverse transcription PCR using a GeXP analyser.

Author information

1
Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, Nanning, Guangxi, China.
2
Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China.

Abstract

OBJECTIVES:

In order to develop a multiplex RT-PCR assay using the GeXP analyser for the simultaneous detection of four different NA serotypes of H5-subtype AIVs, effective to control and reduce H5 subtype of avian influenza outbreak.

DESIGN:

Six pairs of primers were designed using conserved and specific sequences of the AIV subtypes H5, N1, N2, N6 and N8 in GenBank. Each gene-specific primer was fused at the 5' end to a universal sequence to generate six pairs of chimeric primers, and one pair of universal primers was used for RT-PCR, and PCR product separation and detection were performed by capillary electrophoresis using the GenomeLab GeXP genetic analysis system.

SETTING:

Single and mixed avian pathogen cDNA/DNA templates were employed to evaluate the specificity of a multiplex assay with a GeXP analyser. Corresponding specific DNA products were amplified for each gene, revealing amplification peaks for M, H5, N1, N2, N6 and N8 genes from four different NA subtypes of influenza A H5 virus.

SAMPLE:

A total of 180 cloacal swabs were collected from poultry at live bird markets.

MAIN OUTCOME MEASURES:

The multiplex PCR assay demonstrated excellent specificity, with each pair of specific primers generating only products corresponding to the target genes and without cross-amplification with other NA-subtype influenza viruses or other avian pathogens. Using various premixed ssRNAs containing known AIV target genes, the detection limit for the multiplex assay was determined to be 10(2) copies/μl. The GeXP assay was further evaluated using 180 clinical specimens and compared with RRT-PCR (real-time reverse transcriptase PCR) and virus isolation.

CONCLUSIONS:

This GeXP analyser-based multiplex assay for four different NA subtypes of H5 HPAI viruses is both highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples.

KEYWORDS:

Differential diagnoses; GeXP analyser; H5 avian influenza viruses; HA typing; NA typing; multiplex detection

PMID:
26677838
PMCID:
PMC4746555
DOI:
10.1111/irv.12370
[Indexed for MEDLINE]
Free PMC Article

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