JCM Accepts, published online ahead of print on 14 March 2007 J. Clin. Microbiol. doi:10.1128/JCM.02392-06 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Human Influenza A (H5N1) Detection by a Novel Multiplex PCR Typing Method
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Shumei Zou1, Jian Han2, Leying Wen1, Yan Liu3, Kassi Cronin2, Shanjuan H. Lum2, Lu
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Gao2, Jie Dong1, Ye Zhang1, Yuanji Guo1, Yuelong Shu1*
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Running head: Human Influenza A (H5N1) Detection
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Control and Prevention, State Key Laboratory of Infectious Disease Control and
Chinese Center for Disease Control and Prevention, National Institute for Viral Disease
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Prevention, Beijing, China;
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Genaco Biomedical Products, Inc. Huntsville, Alabama, U.S.A
Anhui Provincial Center for Disease Control and Prevention, Hefei, China.
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* Correspondence should be addressed to Dr. Yuelong Shu. Associate Professor at the
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Chinese Center for Disease Control and Prevention, Institute for Viral Disease Control
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and Prevention, China National Influenza Center. Yingxin Jie 100, Xuanwu District,
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Beijing, P.R.China 10052. Tel: +86-10-6357-7499 or at
[email protected]
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ABSTRACT
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We report the use of ResPlex™III for genotyping influenza A viruses. The performance
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characteristics of the assay regarding to H5N1 is further evaluated. The ResPlex™
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system incorporates a novel multiplex PCR technology, Tem-PCR (target enriched
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multiplex PCR), to simultaneously amplify multiple molecular targets in one reaction.
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The ResPlex™III assay targets the H1, H2, H3, H5, H7, H9, N1, and N2 genes from the
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influenza A virus, as well as the NS genes from influenza A (NSA) and B (NSB) viruses,
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providing detection and genotyping of influenza A and B. The analytical sensitivity for
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detecting the H5, N1 and NSA genes were 1, 10-1, and 10 TCID50/200µl/reaction,
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respectively. A total of 217 sequential clinical samples including 14 samples with human
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H5N1 infections were tested by the ResPlex™III and the results were compared to a
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reference standard combined by results of viral culture, conventional reverse transcriptase
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and real-time PCR. The clinical sensitivity and specificity for detecting H5N1 were
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93.3% and 100%, respectively, indicating that different subtypes of influenza A can be
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quickly and correctly identified using the ResPlex™III genotyping approach.
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INTRODUCTION
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Since 1997, the H5N1 subtype of the influenza A virus has been circulating in Asia, and
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now in European countries, causing widespread infection in multiple avian species. So far,
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human-to-human transmission has been suspected in only one case. If an outbreak of
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H5N1 occurs in humans, millions of people worldwide may die from the pandemic.
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Limited amounts of effective vaccines and drug treatments make the prevention and
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control of this disease difficult. Thus, the most effective measure for containing a
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potential outbreak is a large-scale quarantine of suspected patients.
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To minimize the social and economic impact of a large-scale quarantine measure, a quick,
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accurate and comprehensive diagnostic system is essential. Current diagnostic methods,
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such as culture, immunoassays and PCR based molecular tests, lack the ability to meet
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this challenge. We believe that an ideal diagnostic test for avian influenza pandemic
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surveillance and outbreak control should satisfy the following requirements: 1) High
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throughput: allows the analysis of hundreds, even thousands of samples per day; 2)
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Multiplex: detects multiple relevant molecular targets using only one patient sample,
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conducting one experiment, in one reaction system; 3) Safety: minimizes the risk to
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healthcare providers by inactivating pathogens at the beginning of the test procedure; 4)
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Accuracy: provides validated assay specificity and sensitivity; 5) Speed: the entire
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procedure, from sample collection to result output, takes less than 5 hours; 6) Ease of use:
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automates assay procedures making it possible to quickly establish a surveillance
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laboratory network without extensive training. The US Food and Drug Administration
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has approved a real-time PCR assay for the identification of influenza A/H5 (Asia
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lineage). This diagnostic test, developed by the United States Centers of Disease Control
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(CDC), was intended to be used in public health laboratory networks for surveillance of
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H5N1 outbreaks (2). In this study, we report the development of a ResPlex ™ III
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multiplex PCR assay for influenza A typing analysis. The analytical and clinical
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sensitivity and specificity of the assay were evaluated regarding to the H5N1(Asian
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lineage) virus.
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MATERIALS AND METHODS
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Patient Samples and Viral Isolates. One important function of our National Influenza
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Center at the Chinese Centers for Disease Control is to monitor the outbreaks of influenza
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by collaborating with established provincial surveillance laboratories and testing samples
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submitted to us from the provincial laboratories. From October 2005 to February of 2006,
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Chinese CDC received total of 217 human respiratory samples from 16 provinces (AnHui,
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Liaoning, XinJiang, GuangXi, HeBei, JiangXi, FuJIan, XiZang, HuNan, ShanDong,
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SiChuan, JiLin, GuiZhou, HuBei, and GuangDong) to rule out avian influenza (H5N1).
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Majority of clinical samples were pharyngeal swabs (83%) or tracheal aspirates (17%).
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Samples were collected in 0.9% NaCl and shipped to Beijing at 4ºC via express mail
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within 24 hours of collecting. If not shipped immediately, samples were kept frozen at or
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below -70ºC until shipping. Upon arrival, if culture is setting up immediately, the
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samples are stored at 4ºC, otherwise, samples were stored at or below -70ºC. Viral
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isolation and culture were performed following WHO guidelines (WHO/CDS/CSR/NCS/2002.5
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Rev. 1). All
samples were investigated with multiple methods, including viral culture, RT-
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PCR, and real-time PCR.
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One human H5N1viral isolate, A/Anhui/1/2005, was used for analytical sensitivity
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studies. The TCID50 for this isolate was determined using standard viral culture method
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(4). Briefly, viral isolate harvested from chicken embryo culture were serial diluted in
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quadruplicate and add onto MDCK (adin-Darby canine kidney) cell monolayer in log
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phase growth. The TCID50 (50% Tissue Culture Infectious Doses) is determined
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following a method described by the Reed-Muench(8).
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Determination of analytical sensitivity. The viral isolate (A/Anhui/1/2005) with known
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TCID50 was serial diluted in PBS. At each concentration, three samples were prepared.
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Nucleic acid isolation was performed on each sample and then subject to amplification
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with ResPlex™ III. The analytical sensitivity is determined as the lowest TCID50 at
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which all three duplicated samples were successfully detected.
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Nucleic Acid Isolation and Amplification with RT-PCR and Real Time PCR.
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Qiagen QIAamp Viral RNA mini kit (Valencia, CA) was used for the extraction of
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nucleic acid from all of the samples. The starting material for nucleic acid isolation were
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200 µl respiratory sample (in 0.9% NaCl) or 200 µl viral isolate in dilution buffer (PBS).
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RNA was eluted into 50µl water.
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Reference standard set up. Even though the ResPlex™III can provide genotyping
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information for many influenza A strains, the most important intended use of this assay is
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to identify influenza A, H5N1 (Asia lineage) as a confirmatory test. To evaluate the
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performance of ResPlex™III in this regard, a reference standard was established to
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represent the combined results of the viral culture, RT-PCR, and real-time PCR. A
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sample was determined to be positive for H5N1 when at least two of the three reference
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methods were positive. For ResPlex™III, a sample was determined to be positive for
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H5N1 only when all three targets (H5, N1 and NSA) were positive.
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Conventional reverse transcriptase PCR (RT-PCR) and Real-time PCR. We have
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reported the development of reverse transcriptase PCR and real time PCR assays for
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detecting H5N1 from human clinical specimens (9). The primer sequences for these
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studies were listed below:
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RT PCR Primers
Real-Time PCR primers
H5HA-F GCCATTCCACAACATACACCC
FluA-F GACCRATCCTGTCACCTCTGAC
H5HA-R CTCCCCTGCTCATTGCTATG
FluA-R GGGCATTYTGGACAAAKCGTCTACG
N1-F
TTGCTTGGTCAGCAAGTGC
FluA-p TGCAGTCCTCGCTCACTGGGCACG
N1-R
CAGTCACACCATTTGGATCC
H5HA-F TGGAAAGTGTAARAAACGGAACGT H5HA-R TGATTGCCAGYGCTAGGGAACT H5HA-p TGACTACCCGCAGTATTCAGAAGAAGCAAGACTAA
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Amplification and Detection with Tem-PCR. The analytical sensitivity of ResPlex™
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III was evaluated with viral isolate strain, A/Anhui/1/2005, which was established from a
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patient sample from Anhui province (8). The TCID50 were determined as described
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previously (4). Multiplex target amplification was carried out using the ResPlex™III
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assay system from Genaco (Huntsville, AL, Cat#011-03). The SuperPrimers™ from the
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assay system contain a mixture of specific primers capable of amplifying the
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Hemagglutinin gene from subtypes H1, H2, H3, H5, H7, and H9, as well as the
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Neuraminidase gene from the N1 and N2 subtypes. Amplification targets for the NS gene
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of influenza types A and B are also included in the mixture. The Qiagen OneStep RT-
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PCR kit (Valencia, CA) was the enzyme and buffer system used for amplification. To set
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up the amplification reaction, 5-20µl RNA template was mixed with 2µl RT-PCR
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Enzyme, 6µl ResPlex™III SuperPrimers, 2µl dNTP mix, 10µl 5X RT-PCR buffer, 5-10
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units of Ribonuclease Inhibitor, and RNAse-free water to bring the final volume to 50µl.
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Reverse transcription and amplification were carried out in a one-step reaction, with a
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GeneAmp® PCR system 9700 (Applied Biosystems), using the specific cycling profile
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described in the Genaco protocol. A non-target amplification control was included in
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each experiment.
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After PCR amplification, 5µl PCR product was directly mixed with 35µl of Genaco
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Detection Buffer and 10µl of Genaco ResPlex™III Bead Mix. Hybridization was carried
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out at 52°C for 10 minutes. For detection, 10µl of diluted Streptavidin-PE was added
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followed by an additional 5 minute incubation at 52°C. Finally, 120µl of pre-warmed
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Genaco Stopping Buffer was added to the sample prior to detection on a Luminex
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instrument. Luminex xMAP technology has been described elsewhere (3).
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RESULTS
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Figure 1 shows representative results of ResPlex™ III analysis. Each column represents a
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specific target and each row represents a sample. The first 20 samples (No. 1-20) were
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clinical samples, including 14 human H5N1 samples identified by the reference methods.
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Samples No. 21-31 were not clinical samples but viral isolates. These samples were used
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to evaluate the usefulness of ResPlex™ III in detecting other H and N targets. The last
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three samples (Sample No. 32-34) were blanks without any template in the reaction. The
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Median Fluorescent Intensity (MFI) of each target from these three blank samples
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represented the background. The cutoff values for determining the positive signal were
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determined specifically for each target. The cutoffs were calculated as the mean MFI plus
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4 times of standard deviation of the blank samples. Positive results were indicated by
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bold font in Figure 1. Results from the reference methods were listed as “Reference”.
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Table 1 shows the analytical sensitivity of the ResPlex™III assay. Then, the analytical
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sensitivity was determined by using serial diluted viral stocks. As shown in Table 1, the
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analytical sensitivity for H5, N1 and the Influenza A type-specific NS gene are 1 TCID50,
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10-1 TCID50, and 10 TCID50, respectively per reaction mixture.
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Table 2 shows the performance characteristics of the ResPlex™III by comparing it
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against the reference standard. A total of 217 clinical samples were sent to China CDC
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during the study period and were included in the study. Among them, the reference
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methods identified 14 human H5N1 positive samples, while the ResPlex™III identified
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13 as positive for H5N1. The assay’s sensitivity, specificity, positive predictive value
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(PPV) and negative predictive value (NPV) were calculated to be 93.3%, 100%, 100%,
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and 99.5%, respectively.
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DISCUSSIONS
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For the 13 H5N1 positive clinical samples identified by the ResPlex™ III assay (Figure 1
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and Table 2), positive results were also obtained from both the RT-PCR and real time
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PCR assays. Viral isolation, on the other hand, failed in three of the 14 cases. Sample
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CnCDC4 was positive for the N1 and NSA target, but negative for H5. Sequence analysis
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showed a one base pair mutation occurred at the 3’ end of the inside reverse primer for
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the H5 gene. This result suggested that the ResPlex™ III assay is vulnerable for viral
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mutations, even though primers were designed by selecting conserved sequences. To
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reduce false negatives, we need to monitor the viral mutations closely and adding new
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primers and probes to adapt the changes. Alternatively, the ResPlex™ III assay should
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amplify more than one targets from the H5 gene to provide additional assurance and
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reduce false negative results.
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The principle of tem-PCR technology has been described previously (1, 5-6).
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Conventional multiplex PCR assays are difficult to develop because of three primary
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problems: incompatible primer sets, high background amplification, and poor
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reproducibility. The tem-PCR technology developed by Genaco successfully addresses
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these problems improving and expanding the possibilities of multiplex PCR assay
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development.
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For each target in the multiplex PCR, nested gene-specific primers are designed and
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included in the reaction (Fo -- forward out; Fi -- forward in; Ri -- reverse in; and Ro --
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reverse out). These primers are used at extremely low concentrations and are used only to
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enrich the targets during the first few cycles of PCR. The inside gene-specific primers
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have tag sequences that can be recognized by a universal set of primers, called
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SuperPrimers™. Only the SuperPrimers™ are included at a concentration necessary for
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exponential amplification, and only the reverse SuperPrimer™ is labeled with biotin.
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Labeled PCR products are detected with a complimentary capture probe that is covalently
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coupled to a color-coded bead. The concentration of the reverse SuperPrimer™ is higher
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than that of the forward SuperPrimer™, the asymmetric PCR yields more reverse strand
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for detection. This also eliminated the need to denature PCR products prior to
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hybridization.
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Tem-PCR works because it addresses three of the most difficult problems inherent in
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multiplex PCR development. First, in a conventional multiplex PCR reaction, each
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primer may require a different optimal annealing temperature or buffer formula. When
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the number of targets increases, it forces all of the primer sets to work under a single
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amplification profile causing multiplex PCR to become difficult under these standardized
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conditions. With tem-PCR, during enrichment stage, nested primers are used for each
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target. This design gives rise to four possible forward and reverse primer combinations
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for amplification. Each combination may have its own optimum amplification profile, but
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given four amplification opportunities, a common condition satisfying all targets can be
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attained.
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Second, conventional multiplex PCR uses multiple sets of high-concentration, labeled
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primers. These primers can associate with one another to form dimers or generate non-
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specific background amplification. Reduced amplification efficiency can also occur
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because excess primers occupy active sites on the polymerase molecule. In addition,
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unused, labeled primers produce background signal and use up reagents during the
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detection portion of the assay. Because of these issues, post-PCR clean up (such as spin
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column purification) is often required to remove these labeled primers before the
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products can be used as probes. These problems are unnecessary because high-
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concentration primers are only required in the last cycles of a PCR reaction. With tem-
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PCR, the amount of gene-specific primers used is only enough to “enrich” the targets and
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incorporate the SuperPrimer™ tag into the products. After enrichment and tag
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incorporation, exponential amplification is carried out with only one pair of primers.
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Because only one primer is labeled, the background is low; therefore, no post-PCR clean
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up is required. The tem-PCR reaction is also very specific and sensitive. No post-
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hybridization washes are necessary. These features make it feasible to fully automate
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laboratory procedures and perform high-throughput clinical studies.
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Finally, a conventional multiplex PCR product is difficult to produce. Maintain the
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quality of all biotin labeled primers in a mix is a difficult task. Lot-to-lot variations often
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limit the performance of a multiplex reaction and require repeated optimization and
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adjustment. With tem-PCR, only small amount of target specific primers are used, and
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only one biotin labeled primer is included, therefore, lot-to-lot variations are small and
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quality control is more manageable.
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The ResPlex™ III assay uses the Luminex instrument for detecting PCR products. The
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hybridization step requires the opening of PCR tubes and mixing PCR products with the
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beads mix. This step is prone to carry-over contamination and strict preventive
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procedures need to be established.
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We have evaluated the performance of the ResPlex™ III assay using viral isolates and
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clinical samples. The assay is very easy to use and rapid. Up to 96 samples can be
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processed together. The test turnaround time is about 5 hours, starting from sample
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preparation to obtaining the results, and the hands-on time less than one hour. We believe
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the ResPlex™ III assay can be used as a medium throughput confirmative test for those
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suspected as H5N1 (Asia lineage).
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ACKNOWLEDGMENTS
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This work was supported by Chinese National Nature and Science Foundation key
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program grant (30599433), and the authors are in debt grateful for support from the
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Chinese influenza/human avian influenza surveillance network.
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