Comparative analysis of different TaqMan real-time RT-PCR assays for the detection of swine Hepatitis E virus and integration of Feline calicivirus as internal control

Ward, Pierre; Poitras, Élyse; Leblanc, Danielle; Letellier, Ann; Brassard, Julie; Plante, Daniel; Houde, Alain

doi:10.1111/j.1365-2672.2008.04104.x

Cited by 51 publications

(58 citation statements)

References 43 publications

(137 reference statements)

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“…The RT-PCR based on ORF2 rarely detected the 100-fold-diluted genotype 3c and 3e samples, and our assays of several clinical plasma samples showed that the RT-PCR based on ORF2 significantly underestimated the HEV RNA concentration in genotype 3c samples compared to the genotype 3f samples. These data agree well with findings described previously by Ward et al (46), who studied the performances of 4 real-time RT-PCRs based on ORF3 and ORF2 by testing Canadian swine genotype 3 samples. Those authors compared the threshold cycle (C T ) values obtained with the real-time RT-PCR tests and found that the RT-PCR test based on ORF3 was the most sensitive test for the detection of swine HEV strains (46).…”

Section: Discussionsupporting

confidence: 83%

“…These data agree well with findings described previously by Ward et al (46), who studied the performances of 4 real-time RT-PCRs based on ORF3 and ORF2 by testing Canadian swine genotype 3 samples. Those authors compared the threshold cycle (C T ) values obtained with the real-time RT-PCR tests and found that the RT-PCR test based on ORF3 was the most sensitive test for the detection of swine HEV strains (46). Unfortunately, those researchers did not determine the HEV subtypes, and their assays were only semiquantitative.…”

Section: Discussionsupporting

confidence: 83%

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Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

et al. 2012

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Genotype 3 hepatitis E viruses (HEVs) are distributed across the world and are now considered to be an emerging public health concern in industrialized countries. At least 10 genotype 3 subtypes have been identified in humans and animals worldwide. It was recently reported that the sensitivities of HEV RNA assays differ greatly. We have assessed the influence of genotype 3 diversity on the performances of two HEV RNA assays: one targeting the ORF3 gene and the other targeting the ORF2 gene. We tested a panel of 5 HEV-positive reference samples of genotypes 3a, 3b, 3c, 3e, and 3f at 10-fold serial dilutions. The HEV RNA concentrations obtained with both reverse transcription (RT)-PCRs were correlated, but the RT-PCR based on ORF2 underestimated the HEV RNA concentrations. The mean [ORF3 ؊ ORF2] difference was 1.41 log copies/ml. We also tested 34 clinical specimens of genotypes 3c (n ‫؍‬ 15), 3e (n ‫؍‬ 4), and 3f (n ‫؍‬ 15), representing the most prevalent subtypes in Europe. The mean [ORF3 ؊ ORF2] differences were 1.41 log copies/ml for genotype 3c, 0.96 log copies/ml for genotype 3e, and 0.70 log copies/ml for genotype 3f. The bias between the 2 RT-PCR assays was significantly greater for genotype 3c than for genotype 3f (P ‫؍‬ 0.007). We therefore recommend the use of an RT-PCR protocol based on ORF3 to quantify HEV RNA of genotype 3 strains.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionsupporting

confidence: 83%

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

et al. 2012

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“…There was no change in the clinical status occurred during the follow-up period (median follow-up time of 33.8 months) in the disease-free group. The comparative C T method used for quantifying the mRNA of onfFN in this study is an acceptable technique (Chang et al, 2002;Ward et al, 2009). The between-run and within-run variations were less than 2%, suggesting reliability of the procedure.…”

Section: Discussionmentioning

confidence: 96%

Serum Oncofetal Fibronectin (onfFN) mRNA in Differentiated Thyroid Carcinoma (DTC): Large Overlap between Disease-Free and Metastatic Patients

Sritara¹,

Charoenphun²,

Ponglikitmongkol³

et al. 2012

Asian Pacific Journal of Cancer Prevention

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Aim: This study assessed if onfFN mRNA in the peripheral blood of patients with DTC can identify individuals with metastatic disease. Methods: Comparison of onfFN mRNA was made among 3 groups: disease-free, lymph node metastasis, and distant metastasis using real-time RT-PCR on 5 ml blood samples from each DTC patient. Results: Fifty-one patients were included: 30 (59%) were disease-free; 7 (13.7%) had lymph node metastasis; and 14 (27.5%) had distant metastasis. OnfFN mRNA levels in the 3 groups were significantly different (P=0.001) but with a large overlap and the expression being highest in the disease-free group. Subgroup analysis of the metastatic groups did not show any effect of age, cell type, and serum TSH, Tg, and antiTg on onfFN mRNA. The within-run and between-run root mean square coefficients of variations were <2%. Conclusion: OnfFN mRNA in patients with DTC cannot identify those with metastatic disease.

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“…Comparisons of RT-PCR assays have shown a 10-to 1,000-fold variation in sensitivity when samples were tested in parallel in the same laboratory (20,21). In a blinded study to investigate the performance of conventional and real-time RT-PCR assays in 20 laboratories that performed HEV RNA detection on a regular basis, variations in sensitivity on the order of 100-to 1,000-fold were found using a standard panel of HEV genotype 3 and 4 strains (12).…”

mentioning

confidence: 99%

Comparison of Real-Time Reverse Transcriptase PCR Assays for Detection of Swine Hepatitis E Virus in Fecal Samples

et al. 2014

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dHepatitis E virus (HEV) is a major cause of acute viral hepatitis in people in many developing countries and is also endemic in many industrialized countries. Mammalian HEV (mHEV) isolates can be divided into at least four recognized major genotypes. Several nucleic acid amplification techniques have been developed for mHEV detection, with great differences in sensitivity. The aim of this study was to compare the performances of two singleplex real-time reverse transcriptase (RT) PCR assays for broad detection of all four mHEV genotypes (assays A and B) and two duplex real-time RT-PCR assays for detection and differentiation of mHEV genotypes 3 and 4 (assays C and D). RNAs extracted from 28 fecal samples from pigs experimentally inoculated with HEV genotype 3 and 186 fecal samples from commercial pigs with unknown HEV exposure were tested by all four assays. In experimental samples, HEV RNA was detected in 96.4% (assay A), 39.2% (assay B), 14.2% (assay C), and 0% (assay D) of the samples. In field samples with unknown HEV exposure, HEV RNA was detected in 67.2% (assay A), 36.4% (assay B), 1.1% (assay C), and 0.5% (assay D) of the samples. The assays showed overall poor agreement ( ‫؍‬ 0.19 to 0.03), with differences in detection rates between assays (P < 0.01). Assays A and B, which broadly detect HEV genotypes 1 to 4, had significantly higher detection rates for HEV RNA than the duplex assays C and D, which were both designed to detect and differentiate between HEV genotypes 3 and 4.

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Comparative analysis of different TaqMan real-time RT-PCR assays for the detection of swine Hepatitis E virus and integration of Feline calicivirus as internal control

Cited by 51 publications

References 43 publications

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

Serum Oncofetal Fibronectin (onfFN) mRNA in Differentiated Thyroid Carcinoma (DTC): Large Overlap between Disease-Free and Metastatic Patients

Comparison of Real-Time Reverse Transcriptase PCR Assays for Detection of Swine Hepatitis E Virus in Fecal Samples

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