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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REFERENCES:
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1. Brunstein J., and E. Thomas. 2006. Direct screening of clinical specimens for
22
multiple respiratory pathogens using the Genaco respiratory panels I & II. Diagn
23
Mol Pathol 15: 169-173.
12
1
2. Centers for Disease Control and Prevention (CDC). 2006. New laboratory
2
assay for diagnostic testing of avian influenza A/H5 (Asian Lineage). Morb
3
Mortal Wkly Rep. 55: 127.
4
3. Dunbar SA., C.A. Vander Zee, K. G. Oliver, K. L. Karem, and J. W. Jacobson.
5
2003. Quantitative, multiplexed detection of bacterial pathogens: DNA and
6
protein applications of the Luminex LabMap™ system. J Microbiol Methods.
7
53:245-252.
8 9 10
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T P
4. Guo YJ and XW Cheng. Laboratory Techniques for Influenza Studies. 1997. Beijing. China SanXia Publishing.
E C
5. Han J. Molecular differential diagnoses of infectious diseases: is the future now?
11
2006. In: Stratton C. and Tang YW. Eds. Advanced Technologies in Diagnostic
12
Microbiology. New York. NY. Springer Publishing Company.
13 14 15 16 17 18
C A
6. Han, J., D.C., Swan, S. J. Smith, S. H. Lum, S.E. Sefers, E. R. Unger, and Y. W. Tang. 2006. Simultaneous amplification and identification of 25 human papillomavirus types with Templex technology. J Clin Microbiol 44: 4157-4162.
7. Reed LJ and H Muench. 1938. A simple method of estimating fifty percent endpoints. Amer. J. Hyg. 27: 493-497.
8. Shu YL., H.J. Yu, and D.X. Li. 2006. Lethal Avian Influenza A (H5N1)
19
Infection in a Pregnant Women in Anhui Province, China. N Engl J Med. 354:
20
1421-1422.
21 22
9. Wen LY, H. Xu, Y. Lan, X. Zhao, X.G. Zhang, D. Y. Wang, L. H. Yao, J. Dong, J. H. Zhang, Y. J. Guo, and Y. L. Shu. 2006. Development of methods
13
1
for detection of H5N1 from human clinical specimens. Zhonghua Shi Yan He Lin
2
Chuang Bing Du Xue Za Zhi. 20: 24-26.
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