Comparison of RNA Extraction and Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Detection of <i>Porcine Reproductive and Respiratory Syndrome Virus</i> in Porcine Oral Fluid Specimens

Chittick, Wayne; Stensland, Wendy R.; Prickett, John; Strait, Erin L.; Harmon, Karen M.; Yoon, Kyoung‐Jin; Wang, Chong; Zimmerman, Jeffrey J.

doi:10.1177/104063871102300208

Cited by 51 publications

(33 citation statements)

References 11 publications

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“…Oral fluid matrix is known to contain inhibitors that can cause reaction failure or can reduce analytical sensitivity (25). Although an optimized extraction protocol to overcome this was used in addition to the use of a double amount of the recommended PCR enzymes for the AB test (25), due to the limited amount of this reagent in the TC and the AD reagents, this strategy could not be used for these two assays.…”

Section: Discussionmentioning

confidence: 99%

“…Although an optimized extraction protocol to overcome this was used in addition to the use of a double amount of the recommended PCR enzymes for the AB test (25), due to the limited amount of this reagent in the TC and the AD reagents, this strategy could not be used for these two assays. This could explain the clear difference in detection rates between the assays used in this study, in which the AB assay presented the highest detection rates on oral fluids using the optimized PCR protocol while the detection rates were lowest for the TC test.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

et al. 2013

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The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) realtime reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days ؊2, P orcine reproductive and respiratory syndrome virus (PRRSV) continues to be the most important pathogen affecting pigs in North America (1). PRRSV is a small, enveloped, single-stranded positive-sense RNA virus of the family Arteriviridae and can be divided into two genotypes: type 1 (European type [EU]) and type 2 (North American type [NA]) (2). Although a variety of commercial vaccines are available on the global market, the virus remains difficult to control, and the demand for PRRSV-naïve replacement genetics and, with it, the need for highly sensitive and specific assays that can detect genetically diverse strains and provide information on the most appropriate samples for testing continue to grow.Presently, many boar studs in the United States are PRRSV negative and are routinely tested for PRRSV to ensure that PRRSV-free semen is used in breeding herds for artificial insemination (1). If previously negative boar studs become infected with PRRSV, it is critical to detect the virus as soon as possible so that any shipments of possible PRRSV-contaminated semen can be stopped.The reverse transcription-PCR (RT-PCR) method in the realtime format is one of the most commonly used techniques for detection of PRRSV RNA because of its sensitivity and specificity and relatively short test turnaround time. However, the high mutation rate, rapid evolution, and genetic variability of PRRSV strains complicate the development of long-term reliable diagnostic assays, and consequently cases of false-negative results with commercially available assays have been reported (3-5).Active PRRSV surveillance in boar studs relies mainly on collection and testing of serum, semen, blood swabs, and, more recently, oral fluids (6, 7). The choice of sample type should take into consideration the availability and ease of collection in addition to the sensitivity and specificity of the RT-PCR assay. Studies have shown that PRRSV RNA can be detected in boar serum, oral fluids, and blood swabs as early as 24 to 48 h postinfection and in semen samples as early as 48 to 120 h postinfection (6-9). Isolatespecific differences in the levels of PRRSV replication and shedding in the host have been reported (10), and as veterinarians and diagnosticians consider using alternative sampling methods, it is important to conduct an unbiased comparison of the ability of different commercial real-time RT-PCR tests to detect genetically diverse isolates of PRRSV in new and conventional sample types.The sample collection ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

et al. 2013

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“…This procedure, which could have produced more accurate results, was abandoned after a few attempts because it caused a great amount of discomfort for the animal and increased the chance of sample contamination. The presence of 20.5-31.9% inconclusive PCR tests on saliva, rectal, and fecal samples (Table 4) is indicative of a low number of viruses, or the presence of inhibitors such as bile salts and polysaccharides in feces 32 and polysaccharides in saliva, 12 which could impact PCR amplification by impeding cell lysis, causing degradation of target DNA or primers, or inactivating the polymerase enzyme, 35 which was not removed by the DNA extraction kit. Observing faint PCR amplifications in the rectal swab of 1 control mink and in the feces sample of another indicates the possibility of sample contamination in an infected environment.…”

Section: Discussionmentioning

confidence: 99%

Detection of Aleutian mink disease virus DNA and antiviral antibodies in American mink (Neovison vison) 10 days postinoculation

2015

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Abstract. Early detection of infection by the Aleutian mink disease virus (AMDV; Carnivore amdoparvovirus 1) by polymerase chain reaction (PCR) or counterimmunoelectrophoresis (CIEP) has important ramifications in virus eradication programs. A spleen homogenate containing a local isolate of AMDV was injected intraperitoneally into black (n = 44) and sapphire (n = 12) American mink (Neovison vison). Animals were euthanized 10 days postinoculation and anti-AMDV antibodies and AMDV DNA were tested in plasma and 7 organs by CIEP and PCR, respectively. Viral DNA was detected in the plasma, spleen, lymph nodes, bone marrow, and lung samples of all inoculated mink, but was not detected in some small intestine, kidney, and liver samples. In contrast, antibodies were detected in the plasma of 3 sapphire (25.0%) and 19 black (43.2%) mink but not in any of the organs. The sensitivity of the CIEP test on plasma samples was 39.3%, implying that low levels of antibodies during the early stages of virus exposure resulted in failure to detect infection by the CIEP test. We concluded that CIEP is not a reliable test for early detection of AMDV infection in mink and that there were considerable differences among mink of each color type for production of detectable levels of antibodies. PCR tests on samples of saliva, rectal swabs, and feces did not produce consistent and reliable results.

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“…The use of oral fluid PCRs was supported by research demonstrating their application to the oral fluid matrix. 1,5,[9][10][11][12] Routine implementation of the PRRSV oral fluid Ig ELISA test in diagnostic laboratories was supported by extensive assessment of assay performance. Specifically, an interlaboratory study of the ELISA using 276 oral fluid samples found 97.5% agreement among 12 participating laboratories.…”

Section: Discussionmentioning

confidence: 99%

“…1 The lysis/binding solution for protocol A2 was prepared by adding 623 μl of carrier RNA to 40 ml of lysis/binding solution without the addition of isopropanol. For the lysis step, 300 μl of sample was added to 450 μl of lysis/binding solution, vortexed for 3 min, and centrifuged at 16,000 × g for 2 min.…”

Section: Diagnostic Assaysmentioning

confidence: 99%

Probability of detecting Porcine reproductive and respiratory syndrome virus infection using pen-based swine oral fluid specimens as a function of within-pen prevalence

et al. 2013

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Pen-based oral fluid sampling has proven to be an efficient method for surveillance of infectious diseases in swine populations. To better interpret diagnostic results, the performance of oral fluid assays (antibody- and nucleic acid-based) must be established for pen-based oral fluid samples. Therefore, the objective of the current study was to determine the probability of detecting Porcine reproductive and respiratory syndrome virus (PRRSV) infection in pen-based oral fluid samples from pens of known PRRSV prevalence. In 1 commercial swine barn, 25 pens were assigned to 1 of 5 levels of PRRSV prevalence (0%, 4%, 12%, 20%, or 36%) by placing a fixed number (0, 1, 3, 5, or 9) of PRRSV-positive pigs (14 days post PRRSV modified live virus vaccination) in each pen. Prior to placement of the vaccinated pigs, 1 oral fluid sample was collected from each pen. Thereafter, 5 oral fluid samples were collected from each pen, for a total of 150 samples. To confirm individual pig PRRSV status, serum samples from the PRRSV-negative pigs ( n = 535) and the PRRSV vaccinated pigs ( n = 90) were tested for PRRSV antibodies and PRRSV RNA. The 150 pen-based oral fluid samples were assayed for PRRSV antibody and PRRSV RNA at 6 laboratories. Among the 100 samples from pens containing ≥1 positive pig (≥4% prevalence) and tested at the 6 laboratories, the mean positivity was 62% for PRRSV RNA and 61% for PRRSV antibody. These results support the use of pen-based oral fluid sampling for PRRSV surveillance in commercial pig populations.

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Comparison of RNA Extraction and Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Detection of Porcine Reproductive and Respiratory Syndrome Virus in Porcine Oral Fluid Specimens

Cited by 51 publications

References 11 publications

Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

Detection of Aleutian mink disease virus DNA and antiviral antibodies in American mink (Neovison vison) 10 days postinoculation

Probability of detecting Porcine reproductive and respiratory syndrome virus infection using pen-based swine oral fluid specimens as a function of within-pen prevalence

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