Real-Time RT-PCR for Detection of Identification of Flu Types A & B and Subtypes H5 & N1

[sharon sanders]

A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1

Chunli Wu^a, Xiaowen Cheng^b, Jianfan He^b, Xing Lv^b, Jingwen Wang^a, Riqiang Deng^a, Qingxing Long^a and Xunzhang Wang^a^,^,
^aState Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China
^bCenters for Disease Control and Prevention, Shenzhen 518020, China
Received 23 May 2007; revised 22 October 2007; accepted 26 October 2007. Available online 19 December 2007.


Abstract

A multiplex real-time RT-PCR method for the simultaneous detection of influenza virus types A and B and identification of subtypes H5 and N1 in a single tube is described. The method was developed with four sets of primers and probes which were specific to influenza virus (sub)types A, B, H5, and N1, and evaluated by using a total of 40 influenza virus reference strains, including 17 avian influenza A (12 H5N1, 1 H1N1, 1 H3N2, 1 H4N6, 1 H7N3, and 1 H9N2), 18 human influenza A (11 H3N2, 6 H1N1 and 1 H5N1) and 5 influenza B viruses. The method exhibited a high specificity and sensitivity of approximately 10¹?10² copies/μl for each (sub)type and a high reproducibility with intra- and inter-assay CV from 0.13 to 4.24%. In an analysis of 189 clinical samples from patients during the year 2004 and 2005, the method identified 81 positive samples (42.9%) and identified simultaneously 14 type B samples and 11 subtype N1 samples, in comparison only 46 positive samples (24.3%) identified by the conventional culturing method. The method would be a useful molecular diagnostic tool for large-scale screening of clinical samples for influenza virus.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T96-4RCW54S-1&_user=10&_coverDate=03%2F31%2F2008&_rdoc=12&_fmt=summary&_orig=browse&_srch=doc-info(%23toc%235106%232008%23998519998%23680833%23FLA%23display%23Volume)&_cdi=5106&_sort=d&_docanchor=&_ct=45&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=9e27c99e3cecef26356b50c36cc8e731

[Anne]

Re: Real-Time RT-PCR for Detection of Identification of Flu Types A & B and Subtypes H5 & N1

http://www.blackwell-synergy.com/doi...X.2007.02253.x





Rapid detection of H5 avian influenza virus by TaqMan-MGB real-time RT-PCR

• Y.Y. Lu,
• J.Y. Yan,
• Y. Feng,
• C.P. Xu,
• W. Shi and
• H.Y. Mao

• <address>Institute of Virology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China</address>

<address class="author_address">Y.Y. Lu, Institute of Virology, Zhejiang Provincial Center for Disease Control and Prevention, Laozhedazhi Road 17#, Hangzhou 310009, China.
E-mail: luyiyuzjh@yahoo.com.cn</address>Abstract


Aims: Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on a TaqMan-minor groove binder (MGB) probe was developed for the rapid detection of avian influenza virus subtype H5.
Methods and Results: Conserved regions in the haemagglutinin genes of avian influenza viruses subtype H5 served as targets for the primers and TaqMan-MGB probe design. Concentrations of primers and probe were optimized to improve the sensitivity and specificity of the reactions. A plasmid containing the haemagglutinin gene was constructed and in vitro transcribed for a quantitative assay of copy numbers of the target gene. The results revealed that the optimal concentration of primers and probe was 640 and 480 nmol l⁻¹, respectively. The threshold of 100 copies of target molecules could be detected. The linear range for detection was determined as 10² to 10⁸ molecules in reaction.
Conclusions: It took less than 3 h to complete the detection from viral RNA extraction, with good sensitivity and repeatability.
Significance and Impact of the Study: Real-time RT-PCR assay with MGB probe was an effective means for quick and quantitative laboratory detection and monitoring of H5 avian influenza viruses.

[gsgs]

Re: Real-Time RT-PCR for Detection of Identification of Flu Types A &amp; B and Subtypes H5 &amp; N1

how expensive ?

isn't that the most important question in those contexts ?
Still it is rarely mentioned. Why ?