Comparison of Two Commercial Real-Time Reverse Transcriptase Polymerase Chain Reaction Assays for Porcine Reproductive and Respiratory Syndrome Virus
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Brief communication Peer reviewed Comparison of two commercial real-time reverse transcriptase polymerase chain reaction assays for porcine reproductive and respiratory syndrome virus Karen M. Harmon, PhD; Sarah A. Abate, BS; Amy J. Chriswell, BS; Wayne A. Chittick, BS; Erin L. Strait, DVM, P hD Summary Resumen - Comparación de dos pruebas Résumé - Comparaison de deux épreuves Specimens were tested for porcine reproduc- comerciales de reacción en cadena de la commerciales d’amplification en chaîne tive and respiratory syndrome virus by real- polimerasa de transcriptasa reversa en par la polymérase utilisant la transcriptase time reverse transcriptase polymerase chain tiempo real contra el virus del síndrome réverse pour détecter le virus du syndrome reaction (RT-PCR) with two commercial reproductivo y respiratorio porcino reproducteur et respiratoire porcin agent-specific assays. Result discrepancy Se examinaron muestras contra el virus del Des échantillons ont été testés pour le virus between the two tests was confirmed by síndrome reproductivo y respiratorio porcino du syndrome reproducteur et respiratoire conventional RT-PCR, sequencing, or both mediante la prueba de reacción en cadena porcin par réaction d’amplification en chaîne on six of 423 clinical cases. Neither assay de la polimerasa de transcriptasa reversa par la polymérase utilisant la transcriptase detected every positive case. (PCR-RT) en tiempo real con dos pruebas réverse (RT-PCR) avec deux épreuves spéci- comercias agente-específicas. Las discrep- fiques d’agent qui sont disponibles commer- Received: September 28, 2011 ancias entre las dos pruebas se confirmaron cialement. La non-concordance des résultats Accepted: February 27, 2012 mediante el PCR-RT convencional, secuen- entre les deux épreuves a été confirmée par ciación, ó ambos en seis de los 423 casos. RT-PCR conventionnelle, par séquençage, Ninguna de las dos pruebas detectó todos los ou par les deux techniques, pour six des 423 casos positivos. cas cliniques. Aucune des deux épreuves ne détecta tous les cas positifs. orcine reproductive and respiratory region and the primers and probes used both NA and EU subtypes. The previous syndrome virus (PRRSV) is an RNA in real-time PCR can cause false-negative version of the AB reagents (TaqMan NA 3-5 and EU PRRSV reagents) missed or weakly virus causing reproductive failure in results. Most real-time assays include detected some contemporary PRRSV strains Psows and respiratory disease in piglets and one primer pair and a single probe for each (observed and confirmed in Iowa State Uni- growing pigs.1 The high economic impact target, but to enhance the ability to detect a versity Veterinary Diagnostic Laboratory, of this agent dictates the need for rapid and genetically diverse population, it has become more common to utilize multiple primers 2010). The primers and probes were recently accurate diagnosis. Polymerase chain reac- updated to improve the assay’s detection tion (PCR) testing has been increasingly and probes in a single multiplexed assay. In this study, we compared performance of two capability. In the current study, the TC and used as a diagnostic test because of its sensi- updated AB assays were performed on a tivity and specificity and also because of the commercially available PRRSV-specific real- time reverse transcriptase PCR (RT-PCR) subset of clinical specimens submitted to the ability to complete testing the same day the Iowa State University Veterinary Diagnostic assays for detection and differentiation of sample is received. For any PCR test to be Laboratory to compare the performance of North American (NA) and European (EU) effective for determining the absence or pres- the two commercially available tests. ence of the agent of interest, it is essential subtypes of the virus. The assays are manu- that it target a highly conserved region of factured by Applied Biosystems, Foster City, California (AB), and Tetracore, Rockville, Materials and methods the genome. This is somewhat problematic The samples used in this study were ran- with RNA viruses such as PRRSV which Maryland (TC). The VetMax NA and EU PRRSV reagents (AB) and VetAlert NA and domly selected from routine case submis- have high mutation rates, resulting in rapid sions to our laboratory and consisted of 2 EU PRRSV PCR reagents (TC) each con- evolution and genetic variability. Even serum (n = 380), lung tissue (n = 277), oral tain multiple primers and probes to detect minor mismatches between the PCR target fluid (n = 245), environmental samples (n = 8), nasal swabs (n = 7), semen (n = 6), Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, I owa. bronchoalveolar lavage fluid (n = 5), fetal th thoracic fluid (n = 4), and assorted tissues Corresponding author: Dr Karen Harmon, 1600 S 16 Street, Veterinary Diagnostic Laboratory, (n = 2). Except where noted, nucleic acid Ames, IA 50011; Tel: 515-294-1950; Fax: 515-294-3564; E-mail: [email protected] . extraction was performed using a MagMax This article is available online at http://www.aasv.org/shap.html. Viral RNA Isolation Kit (Life Technolo- Harmon KM, Abate SA, Chriswell AJ, et al. Comparison of two commercial real-time reverse gies, Carlsbad, California) in conjunction transcriptase polymerase chain reaction assays for porcine reproductive and respiratory syndrome v irus. with a Kingfisher or Kingfisher 96 J Swine Health Prod. 2012;20(4):184–188. instrument (Thermo Scientific, Waltham, 184 Journal of Swine Health and Production — July and August 2012 Massachusetts), according to the manufac- the discrepant pools were tested if sufficient of 19 of the discrepant cases were subjected turer’s recommendations. For tissue samples, sample was available. Cases that repeated to further testing by both assays, and 10 of a 10% to 20% (w/v) homogenate was as discrepant by real-time RT-PCR were these repeated as discrepant. Of those 10, six prepared using Earle’s balanced salt solution subjected to conventional RT-PCR using were lung samples, two were serum, and two and then processed on high speed for 120 primers specified by Christopher-Hennings were oral fluid. Table 1 summarizes sample seconds (Stomacher 80; Seward, Bohemia, et al7 and the Qiagen OneStep RT-PCR Kit types tested and results. The most common New York). The homogenized sample was (Qiagen Inc, Valencia, California), following observations for those that did not repeat centrifuged at approximately 4200g for 10 the manufacturer’s recommendations. Prim- as discrepant were first, the original test was minutes and the supernatant was used in ers were added at a concentration of 600 nM negative on pooled sera and positive on one the extraction procedure. Extraction of oral each, and the following thermal cycling pro- or more individual samples from the pool fluids, semen, and environmental samples file was used: 50°C for 30 minutes, 95°C for when retested (n = 3), and second, the origi- was performed as described by Chittick 15 minutes, then 35 cycles of 94°C for 30 nal Ct was lower than 36 cycles, which is 6 et al (extraction A1) except the elution seconds, 58°C for 30 seconds, and 72°C for considered weakly positive, and the original buffer volume was reduced to 50 µL. The 1 minute. A final elongation step of 72°C for result did not repeat (n = 5). One sample program used for Kingfisher extraction was 10 minutes was used. Detection of conven- had an original Ct of 28.15 only with the the Standard MagMax program provided tional RT-PCR products was performed on AB test, but upon retest was positive with by Applied Biosystems. Real-time RT-PCR a Qiaxcel (Qiagen, Inc) capillary electropho- both assays. Original and retest results of was performed on nucleic acid extracts of resis system using a DNA screening cartridge these nine cases are summarized in Table 2. a subset of samples submitted to the Iowa and method AM420. Sequencing of open Of the 10 repeating discrepant cases, nine State University Veterinary Diagnostic reading frame 5 (ORF5) was also attempted were positive by the AB assay and negative Laboratory using both VetAlert NA and EU on at least one discrepant sample from these with the TC reagents. The remaining dis- PRRSV PCR reagents (TC) and VetMax cases using forward primer 5'-AAG GTG crepant case was positive by TC and negative NA and EU PRRSV PCR reagents (AB). GTA TTT GGC AAT GTG TC-3' and by the AB assay. Reactions were performed according to reverse primer 5'-GAG GTG ATG AAT The 10 cases that repeated as discrepant were manufacturers’ instructions using the AB TTC CAG GTT TCT A-3' and the qScript subjected to additional PRRSV testing by 7500 Fast instrument. For each group of Custom One-Step RT-PCR Kit (Quanta conventional RT-PCR and ORF5 sequenc- samples, both assays were performed on the Biosciences, Gaithersburg, Maryland). ing attempts. The purpose for sequencing same day from the same nucleic-acid extract. Polymerase chain reaction setup followed was twofold: first, to confirm the presence of For the AB testing, analysis was performed the manufacturer’s recommendations, with PRRSV in the sample, and second, to deter- using the auto baseline setting. Thresholds each primer included at 320 nM and the mine relatedness of the viruses involved. for NA and EU PRRSV were set at 0.10 following cycling conditions: 48°C for 20 Five of the cases positive by the AB test and and 0.05, respectively. For TC, manual minutes, then 94°C for 3 minutes, followed negative by the TC test were confirmed baseline was used for analysis at the instru- by 45 cycles of 94°C for 30 seconds, 50°C PRRSV-positive by conventional RT-PCR. ment’s default setting of three to 15 cycles. for 50 seconds, and 68°C for 50 seconds. Two of the five also yielded complete or Thresholds for NA and EU PRRSV were set The final elongation step was 68°C for 7 partial ORF5 sequences consistent with at 51,000 and 145,000, respectively.