Identification of New Influenza B Virus Variants by Multiplex Reverse Transcription-PCR and the Heteroduplex Mobility Assay

Abstract

A quick genetic approach for the screening of influenza virus variants was developed in this laboratory (S. Zou, J. Clin. Microbiol. 35:2623–2627, 1997). It uses multiplex reverse transcription and multiplex PCR to amplify and differentiate the variable region of the hemagglutinin genes of different types and subtypes of influenza viruses. Variants within the same type or subtype are then identified by the heteroduplex mobility shift assay of the amplicons. The method was used to screen influenza virus isolates received from provincial laboratories during the 1996–1997 season and was able to identify new influenza B virus variants. Sequencing of the amplicons derived from the hemagglutinin gene of the identified variants and comparison with the vaccine strain B/Harbin/7/94 showed substitution rates of 2.26 to 2.55% at the nucleotide level and 4.26 to 4.68% at the amino acid level. The result further demonstrated that the approach provides a quick, sensitive, and reliable screening for influenza virus variants. It also suggested the necessity of close monitoring of influenza B virus isolates in the 1997–1998 season and critical evaluation of the reference strain for the type B influenza virus.

Use of Base Excision Sequence Scanning for Detection of Genetic Variations in St. Louis Encephalitis Virus Isolates

“…Viruses, because of their small genome size and in many cases their RNA content, are not easily typed by such methods as pulsed-field gel electrophoresis of DNA macrorestriction fragments or PCR-based techniques (random amplification of polymorphic DNA, repetitive extragenic palindromic elementbased PCR, enterobacterial repetitive intergenic consensusbased PCR, or PCR-ribotyping). Recently, viruses have been typed by various techniques used as alternatives to DNA sequencing (1,3,5,7,8,12,(17)(18)(19)(20)(21)(23)(24)(25). Most of these involve PCR amplification followed by restriction fragment length polymorphism (RFLP) analysis, single-strand conformational polymorphism (SSCP) analysis, cleavase fragment length polymorphism (CFLP; Third Wave Technologies, Madison, Wis.), and heteroduplex migration analysis (HMA).…”

“…The SSCP and HMA techniques have successfully been used for genotype characterization of hepatitis C virus (11) and for epidemiological studies of parvovirus B19, enterovirus, Epstein-Barr virus, human immunodeficiency virus type 1, influenza virus, and bunyaviruses (1,5,8,12,17,19,25).…”

Endocannabinoid signaling regulates regenerative axon navigation in Caenorhabditis elegans via the GPCRs NPR‐19 and NPR‐32

“…We used the heteroduplex mobility assay (HMA) (Zou et al . ) to detect the presence of short insertions or deletions in the nape‐1 gene caused by nonhomologous end joining following DNA cutting by Cas9. Positive samples that showed a band shift in HMA were selected for DNA sequencing.…”

Coupling multiplex RT-PCR to a gene chip assay for sensitive and semiquantitative detection of severe acute respiratory syndrome-coronavirus

“…To overcome this limitation, the multiplex RT-PCR (MRT-PCR) seems to be a good alternative because it can simultaneously detect multiple target sequence regions of a pathogen genome in one assay. For several respiratory infectious diseases, this method has been reported useful in detecting viruses, 10 such as influenza, [11][12][13] parainfluenza, 14,15 and respiratory syncytial viruses (RSV). 12,15 However, some practical problems, including poor sensitivity and specificity, limit this method as a general diagnostic tool.…”