Detection and differentiation of four poultry diseases using asymmetric reverse transcription polymerase chain reaction in combination with oligonucleotide microarrays.
Asymmetric reverse transcription polymerase chain reaction (RT-PCR) and microarrays were combined to distinguish 4 viruses, including Avian influenza virus (AIV), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), and Infectious bursal disease virus (IBDV), and hemagglutinin (HA) subtypes H5, H7, and H9, and neuraminidase (NA) subtypes N1 and N2 of AIV. The AIV matrix protein (M), and HA and NA genes, IBV nucleoprotein (NP) gene, NDV NP gene, and IBDV A fragment gene were cloned into plasmids. These genes were amplified from these positive recombinant plasmids, which included the inserted target genes by PCR. The PCR products were purified and printed on the amino-modified slides as the probes. RNA was extracted from samples and labeled by asymmetric RT-PCR using a cyanine (Cy)3-labeled primers. The labeled complementary (c)DNA was hybridized to the probes immobilized on the glass slides. After hybridization, the microarrays were scanned, and the hybridization pattern agreed perfectly with the known location of each probe. No cross-hybridization could be detected. Results demonstrated that microarray based on asymmetric RT-PCR is an effective way to distinguish AIV, IBV, NDV, and IBDV simultaneously.
Tao
Qimeng
Q
Animal Influenza Laboratory of the Ministry of Agriculture and State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China.
Wang
Xiurong
X
Bao
Hongmei
H
Wu
Jianan
J
Shi
Lin
L
Li
Yanbing
Y
Qiao
Chuanling
C
Yakovlevich
Samuilenko Anatolij
SA
Mikhaylovna
Poukhova Nina
PN
Chen
Hualan
H
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D006389:Hemagglutinins, Viral / Q000235:genetics; D001351:Infectious bronchitis virus / Q000235:genetics*; D009439:Neuraminidase / Q000235:genetics; D009522:Newcastle disease virus / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009975:Orthomyxoviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011201:Poultry Diseases / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D046749:Starlings / Q000821:virology; D014422:Turkeys / Q000821:virology
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D006389:Hemagglutinins, Viral; D009439:Neuraminidase