A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to 2012

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and β showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and β-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10−5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, β-, and γ-.

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Quantification of viable eggs of the potato cyst nematodes (Globodera spp.) using either trehalose or RNA-specific Real-Time PCR

Nematology ◽

10.1163/15685411-00002848 ◽

2014 ◽

Vol 16 (10) ◽

pp. 1219-1232 ◽

Cited By ~ 6

Author(s):

Johanna E. Beniers ◽

Thomas H. Been ◽

Odette Mendes ◽

Marga P.E. van Gent-Pelzer ◽

Theo A.J. van der Lee

Keyword(s):

Real Time ◽

Visual Inspection ◽

Membrane Integrity ◽

Globodera Pallida ◽

Potato Cyst Nematodes ◽

Original Sample ◽

Rt Pcr ◽

Cyst Nematodes ◽

Pcr Assay ◽

Field Samples

Two novel methods for the quantitative estimation of the number of viable eggs of the potato cyst nematodes (Globodera pallida and G. rostochiensis) were tested and compared with visual inspection. One is based on the loss of membrane integrity upon death and uses trehalose (a disaccharide) as a marker, the second test exploits the rapid degeneration of mRNA upon decease with a RNA-specific Real-Time Polymerase Chain Reaction (RT-PCR) assay. The viability of eggs in suspensions with different numbers of eggs was determined morphologically and was compared with both trehalose and elongation-factor-1-alpha (EF1α) mRNA measurements. The trehalose assay provided results that were close to those of the visual assessment using a microscope but only when samples contained low numbers of eggs. The lowest detectable value is 1.1 egg in the original sample and small differences in the number of viable eggs can be detected. Unfortunately, trehalose measurements reached a saturation limit at 1 cyst 10 μl−1; therefore, samples with nematode numbers above 262 eggs have to be diluted. The presence of dead cysts did not have a negative effect on the trehalose measurements. However, the use of egg suspensions instead of encysted eggs improved both the trehalose absorbance and the reliability of the measurements. When cysts were exposed to a treatment with allylisothiocyanate, the trehalose measurement detected the presence of more viable eggs than a hatching assay. The RT-PCR assay required a minimum of 30 eggs before detection occurred but can detect up to 8000 eggs in a 25 μl sample, which is an advantage when samples with high PCN infestations have to be processed. However, the confidence intervals (CI) of the RT-PCR assay are larger than those of the trehalose assay, which results in a high variation of single measurements. For example, at a density of 210 eggs in the original sample the 95% CI for the trehalose assay covers 191-228 eggs, and the 95% CI for the RT-PCR assay for G. pallida lies between 73 and 602 eggs and for G. rostochiensis between 59 and 745 eggs. Trials with field samples using both methods supported the laboratory tests. 95% of the field samples tested with the trehalose assay lie within the CI of the standard curve compared to 58% of the RT-PCR tested samples for G. pallida. The measurements of the field samples of G. pallida and G. rostochiensis populations using both methods resulted in larger numbers of viable eggs being detected compared to a hatching test. Neither of the investigated methods in their current state of development is optimal for use as a substitute for the visual inspection used in monitoring labs. The variance of the RT-PCR assay is too high if used for quantitative monitoring; the density range of eggs that can be detected using the trehalose assay is too small.

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Development of rapid, sensitive one-tube duplex RT-PCR assay for specific and differential diagnosis of Chikungunya and dengue

Journal of Virological Methods ◽

10.1016/j.jviromet.2013.07.029 ◽

2013 ◽

Vol 193 (2) ◽

pp. 521-524 ◽

Cited By ~ 7

Author(s):

Kallol Saha ◽

Rushna Firdaus ◽

Sekhar Chakrabarti ◽

Provash Chandra Sadhukhan

Keyword(s):

Differential Diagnosis ◽

Rt Pcr ◽

Pcr Assay

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Detection of Seoul virus in wild brown rats (Rattus norvegicus) from pig farms in Northern England

Veterinary Record ◽

10.1136/vr.105249 ◽

2019 ◽

Vol 184 (17) ◽

pp. 525-525 ◽

Cited By ~ 5

Author(s):

Ellen G Murphy ◽

Nicola J Williams ◽

Malcolm Bennett ◽

Daisy Jennings ◽

Julian Chantrey ◽

...

Keyword(s):

Age Differences ◽

Rattus Norvegicus ◽

Sanger Sequencing ◽

Reservoir Host ◽

Rt Pcr ◽

Pcr Assay ◽

Seoul Virus ◽

Pig Farms ◽

Wild Rats ◽

Globally Distributed

IntroductionHantaviruses are maintained by mammalian hosts, such as rodents, and are shed in their excretions. Clinical disease can occur in humans from spillover infection. Brown rats (Rattus norvegicus) are the globally distributed reservoir host of Seoul virus (SEOV). Human cases of SEOV-associated haemorrhagic fever with renal syndrome (SEOV-HFRS)have been reported in Great Britain (GB) since 1977.MethodsBrown rats (n=68) were trapped from a variety of peridomestic locations, with a focus on pig farms. Kidney and lung tissues were tested for viral RNA using a pan-hantavirus RT-PCR assay followed by Sanger sequencing and analysis.ResultsSEOV RNA was detected in 19 per cent (13/68, 95% CI 11 to 30) of rats and all sequences fell within SEOV lineage 9. Twelve sequences were highly similar to each other and to the previously reported GB Humber strain of SEOV (98 per cent). One rat SEOV sequence was more distant. The SEOV prevalence in rats from pig farms was significantly greater (p=0.047) than other sites sampled. No significant sex or age differences were observed among positive and negative rats.DiscussionThe results from this study suggest that SEOV could be widespread in wild rats in GB and therefore pose a potential risk to public health.

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