Annals of Clinical & Laboratory Science 39:155-159 (2009)
© 2009 Association of Clinical Scientists
Validation of the TaqMan Influenza A Detection Kit and a Rapid Automated Total Nucleic Acid Extraction Method to Detect Influenza A Virus in Nasopharyngeal Specimens
Shelly Bolotin1,
Cedric De Lima1,
Kam-Wing Choi1,
Ernesto Lombos1,
Laura Burton1,
Tony Mazzulli1,2,3 and
Steven J. Drews1,2,3
1 Ontario Public Health Laboratories, 2 Department of Microbiology, Mount Sinai Hospital, and 3 Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Address correspondence to Dr. Shelly Bolotin, Ontario Public Health Laboratories, Molecular Diagnostics, 81 Resources Road, Etobiocoke, Ontario M9P 3T1, Canada; tel 416 235 6504; fax 416 235 6550; e-mail shelly.bolotin{at}ontario.ca.
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Abstract
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This study describes the validation of the TaqMan® Influenza A Detection Kit v2.0 combined with an automated nucleic acid extraction method. The limit of detection of this assay was determined by probit regression (95% confidence interval) to be 2 influenza A/PR/8/34 (H1N1) virus particles per microlitre. One hundred and eleven specimens previously tested using the Seeplex® RV assay and viral culture methods were tested using the TaqMan Influenza A Detection Kit. Compared to the aggregate gold-standard, the sensitivity and specificity of the TaqMan Influenza A Detection Kit were 100% (35/35) and 97% (74/76), respectively. Because of its accuracy, quick turn-around-time and lyophilized bead form, the TaqMan Influenza A Detection Kit, combined with the NucliSense® easyMAGTM automated extraction method, constitutes a reliable protocol for influenza A diagnosis.
Keywords: influenza, respiratory viruses, pandemic preparedness, molecular diagnostics
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Introduction
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In addition to the burden of seasonal influenza, the possible threat of pandemic influenza underscores the need for effective influenza detection strategies. Pandemic influenza detection methodologies must be rapid, highly reliable, and high-throughput [1]. The gold-standard method for influenza detection has traditionally been viral isolation [2,3], which has a turn-around-time of up to 14 days [4] and is not sensitive enough to detect influenza in patients with low viral loads [5]. Other options for rapid detection of influenza virus such as rapid antigen testing of specimens have been hampered by poor test characteristics [6]. Methods such as direct fluorescence antibody detection are labor intensive, technically complex, and require a level of subjectivity that may bias test results [6–8]. Due to these issues, in recent years the use of molecular detection methods for influenza has increased.
The pairing of automated extraction methods to real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays has been shown to be an effective means of rapidly detecting influenza A in respiratory specimens [9]. In this study we report the verification of TaqMan Influenza A Detection Kit v2.0 combined with an automated NucliSense easyMAG extraction method. This influenza A detection method was compared to both viral-culture detection and the Seeplex RV detection kit, a multiplexed reverse-transcription-PCR kit for detection of 12 respiratory viruses, including influenza A and B, respiratory syncytial viruses A and B, metapneumovirus, parainfluenza viruses one, two, and three, rhinovirus A, and coronavirus 229E/NL63 and OC43 [10,11].
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Materials and Methods
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Nasopharyngeal swabs from patients with influenza-like illness were collected using the flocculated Starswab Multitrans Collection and Transport System (Starplex, Bolton, ON, Canada). One specimen per patient was used in this study. The patient population was variable and not restricted to any specific demographic among Ontario residents. This study was performed on a waived-consent protocol utilizing residual laboratory specimens.
Upon arrival in the laboratory, specimens were aliquoted for both viral culture and molecular testing. Total nucleic acid was extracted using the NucliSense easyMAG system (bioMérieux, Montreal, QC, Canada) according to the manufacturer’s instructions. To control for extraction, all specimens were tested for human target gapdh by using the gapdh TaqMan control real-time PCR assay (ABI, Foster City, CA, USA). Specimens were then tested using the Seeplex RV Detection kit protocol (Seegene, Inc., Rockville, MD, USA). Endpoint RT-PCR products were visualized using gel electrophoresis with ethidium bromide staining. Specimens were also cultured on a rhesus monkey kidney cell line (Diagnostic Hybrids, Athens, OH, USA) followed by post-culture direct fluorescence antibody (DFA) staining for influenza A and influenza B (Diagnostic Hybrids, Athens, OH, USA). Cultures were kept for 10 days before being called negative for influenza. Influenza positivity was determined based on the aggregate gold-standard of any specimen positive by either the Seeplex RV Detection kit or a culture detection-based method.
Specimens tested using the Seeplex RV Detection Kit and virus culture methods were picked for testing using the TaqMan Influenza A Detection Kit v2.0 (Applied Biosystems, Foster City, CA, USA), including 35 influenza A H3N2 positive isolates and 76 influenza negative isolates. Of the influenza negative isolates, 24 were positive for influenza B using the Seeplex RV or viral culture methods. The remaining 52 specimens were negative for both influenza A and B. Specimens of nucleic acid that were not promptly tested using the TaqMan Influenza A Detection Kit v2.0 were stored at –80°C until use.
The TaqMan Influenza A Detection Kit was used for detection of influenza A RNA according to the manufacturer’s instructions. The target of this kit is a conserved region of the influenza A matrix gene. In addition to DNA polymerase and dNTPs, the detection kit contains reconstitution buffer to dissolve the beads, reverse transcriptase to convert viral RNA to cDNA, internal positive control (IPC) nucleic acid, and primers and TaqMan probes for the detection of both influenza A and the IPC. All runs were performed on the Step One Plus real-time PCR thermocyler (ABI, Foster City, CA, USA) using a one-step RT-PCR format. Cycling parameters were: 1 min at 95°C, 15 min at 60°C, and 5 min at 95°C, followed by 40 cycles of 20 sec at 95°C and 1 min at 60°C. A manual baseline was set from cycles 6–15 and a manual cycle threshold (Ct) was set at 0.2.
Serial 10-fold dilutions of titred influenza A/PR/8/34 (H1N1) virus (Advanced Biotechnologies, Inc.) were used for limit of detection (LOD) studies. The starting concentration of virus was 3x107 viral particles/ml as determined by the manufacturer. Titred virus was extracted using the NucliSense easyMAG system, as above. To determine the LOD, serial dilutions were tested using the TaqMan Influenza Detection Kit both in triplicate to determine intra-assay variability and in singles 3 times to determine reproducibility. The LOD was calculated by probit regression with a 95% confidence interval (95% CI) using SPSS v. 15 (SPSS, Inc., Chicago, IL, USA).
Sensitivity and specificity were calculated by comparing the TaqMan Influenza A Detection Kit against the aggregate gold-standard of positivity for influenza A by either the Seeplex RV detection or culture detection-based method.
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Results
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To determine the minimum number of viral particles (LOD) that could be detected by the TaqMan Influenza Detection Kit and the Seeplex RV Detection Kit, serial 10-fold dilutions of titred influenza A/PR/8/34 with a starting concentration of 3x107 viral particles/ml were tested. Amplificaion plots for the TaqMan Influenza A Detection kit LOD experiments are shown in Fig. 1
. The LOD was calculated using probit regression (95% CI) and found to be 2 influenza A/PR/8/34 (H1N1) virus particles/µl for the TaqMan Influenza Detection Kit and 8 virus particles/µl for the Seeplex RV assay. Intra-assay variability experiments using each serial dilution in triplicate indicated little variation within experiments (Fig. 1). The assay reproducibilitywas assessed by testing titred virus 4 times. This assay was highly reproducible with variations in cycle threshold (Ct) of 0.05–0.26 Cts between runs.
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Fig. 1. Real-Time PCR amplification plot showing serial dilutions of virus for LOD determination. Serial ten-fold dilutions of titred influenza A/PR/8/34 (H1 N1) virus were made with a starting concentration of 3x107 viral particles/ml. Following RNA extraction isolates were tested using the TaqMan Influenza A Detection Kit. The blue curves represent the amplification curves of influenza A RNA and the fainter gray curves represent the amplification curves of the IPC. The LOD of this assay was calculated to be 2 viral particles/µl.
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One hundred and eleven specimens, previously tested using the Seeplex RV assay and viral culture methods, were tested using the TaqMan Influenza A Detection Kit (Table 1). Of the 76 specimens determined to be influenza A-negative by the Seeplex RV assay, all were culture-negative for influenza A. Of these, 24 were culture or Seeplex positive for influenza B, and 52 were influenza negative by both methods. Of the 35 specimens determined to be influenza A-positive by the Seeplex RV assay, 31 were culture-positive and 4 were culture-negative.
Compared to the aggregate gold-standard, the sensitivity and specificity of the TaqMan Influenza A Detection Kit were 100% (35/35) and 97% (74/76), respectively. This kit was specific to influenza A and did not detect influenza B isolates, which represented 24/76 (31.6%) of our influenza A negative specimens.
Two Seeplex test-negative TaqMan test-positive specimens were identified. It should be noted that both of these isolates were "late positives" and had very high Ct values of 38.2 and 39.6 cycles respectively. The real-time PCR amplification plots for these isolates along with positive and negative controls are shown in Figs. 2a and 2b.
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Fig. 2. a (upper plot) and 2b (lower plot). Real-Time PCR amplification plots of 2 discrepant isolates that were negative for influenza A using both the Seeplex RV Detection Kit and virus culture methods, but were positive using the TaqMan Influenza A Detection Kit.
In Fig. 2a, an isolate with a Ct of 38.2 (isolate trace in green) is shown along with the positive control (in brown) and a negative control (in pink).
In Fig. 2b, an isolate with a Ct of 39.6 (isolate trace in blue) is shown along with the positive control (in brown) and a negative control (in pink).
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The rapidity of the methods for influenza A detection was compared. The TaqMan Influenza A Detection Kit had a turn-around-time of 2.5 hr while the Seeplex RV assay was performed in approximately 7 hr. Turn-around-time for detection of influenza A using viral culture was 10 days.
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Discussion
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This validation study indicates that the pairing of the TaqMan Influenza A Detection Kit with the NucliSense easyMAG extraction system allows for rapid, sensitive, and specific detection of influenza A in nasopharyngeal specimens. Rapid detection of influenza A has been shown to be an important tool for the management of antiviral therapy, during outbreak investigations where patient cohorting may be an option, and in situations where test results may be used to rule out influenza A infection and patients may be removed from respiratory precautions [12–14]. Molecular detection methods offer clinicians options that were not available with culture-based methods, which are limited by extended turn-around-times, or rapid antigen detection methods, which are hampered by poor test characteristics [2,4,6]. Real-time RT-PCR methods have benefits over traditional endpoint molecular methods, including rapid turn-aroundtimes, amplification and target detection within the same reaction vessel, and the conservation of human and laboratory resources. Automated influenza extraction methods are essential for routine viral diagnostics as well as for pandemic planning because they conserve human resources, decrease human-based laboratory errors, and limit the exposure of laboratory workers with little or no immunity to new influenza strains to specimens that may contain large viral titres.
This project was initiated with the goal of verifying commercial products that could be used for rapid detection of influenza A during pandemic or outbreak settings. At this time, many laboratories are still utilizing site-developed or "home-brew" molecular assays for real-time detection of influenza A in patient specimens and for their pandemic preparedness protocols. Unfortunately, the diversity of home-brew assays is not conducive to the standardization of pre-analytical, analytical, and post-analytical laboratory protocols between laboratories and across jurisdictions. The problem of poor standardization is exacerbated by the limited number of Food and Drug Administration (FDA) approved, rapid-detection commercial assays for the detection of influenza A, resulting in their limited use in clinical laboratories.
In this study the TaqMan Influenza A Detection Kit was shown to be a highly sensitive and specific method for the detection of influenza A. Its LOD of 2 viral particles/µl was lower than that of the Seeplex RV assay, which had an LOD of 8 viral particles/µl. The LOD of the TaqMan Influenza A Detection Kit is comparable to previously published home-brew real-time PCR assays [15] as well as commercial systems such as the Luminex RVP assay [16]. The convenient bead format and fast-block thermal cycling conditions result in a short turn-around-time of 2.5 hr. This turn-around-time is comparable to home-brew real time PCR assays [17] and in our experience is shorter than the turn-around-times for commercial tests such as the Luminex RVP assay.
Although the TaqMan Influenza A Detection Kit was highly sensitive and specific (100% and 97.5%, respectively), 2 of the isolates tested were influenza A-negative using the Seeplex RV kit and influenza A-positive using the TaqMan Influenza A Detection Kit. Both of these isolates were reported to the clinicians as influenza negative since they were negative using reference methods. The discrepant test results for these 2 isolates are probably due to the lower LOD of the TaqMan Influenza A Detection Kit compared to the Seeplex RV Detection Kit (2 viral particles/µl vs 8 viral particles/µl).
The TaqMan Influenza A Detection Kit does not detect influenza B strains. However, the greater clinical severity of influenza A infections and the high probability that new emergent or pandemic strains of influenza will be influenza A [1] suggests that this kit may still serve an important function in both clinical and public health settings. In the event that influenza A infection is suspected, the high sensitivity of this assay makes it an excellent "rule-out" test. In addition, the convenient bead form of the assay makes it suitable for use during a pandemic, with reduced preparation time compared to liquid-based molecular diagnostics methods.
In conclusion, the TaqMan Influenza A Detection Kit v2.0 combined with an automated nucleic acid extraction system is an effective method for rapid detection of influenza A in nasopharyngeal specimens.
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Abstract
Introduction
