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1.
Bing Du Xue Bao ; 29(5): 488-94, 2013 Sep.
Article in Chinese | MEDLINE | ID: mdl-24386836

ABSTRACT

H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H5N1 Subtype/isolation & purification , Influenza in Birds/virology , Poultry Diseases/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Chickens , Influenza A Virus, H5N1 Subtype/genetics , Influenza in Birds/diagnosis , Poultry Diseases/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Sensitivity and Specificity
2.
Virol Sin ; 27(2): 120-31, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22492003

ABSTRACT

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.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , Influenza A virus/isolation & purification , Influenza in Birds/virology , Multiplex Polymerase Chain Reaction/methods , Animals , Birds , DNA Primers/genetics , High-Throughput Nucleotide Sequencing/instrumentation , Influenza A virus/classification , Influenza A virus/genetics , Influenza in Birds/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods
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