scholarly journals (Non-)Sense of Milk Testing in Small Ruminant Lentivirus Control Programs in Goats. Comparative Analysis of Antibody Detection and Molecular Diagnosis in Blood and Milk

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 3
Author(s):  
Nadjah Radia Adjadj ◽  
Jo Vicca ◽  
Rodolphe Michiels ◽  
Nick De Regge
Keyword(s):  
Molecular Detection ◽  
Small Ruminant ◽  
Quantitative Polymerase Chain Reaction ◽  
Relative Sensitivity ◽  
Antibody Detection ◽  
Preliminary Evaluation ◽  
Analytical Sensitivity ◽  
Control Programs ◽  
Polymerase Chain ◽  
Higher Sensitivity

Small ruminant lentivirus (SRLV) control programs are mainly based on diagnostic tests performed on blood samples collected from sheep and goats. Since blood sampling is costly and stressful for the animals, we evaluated whether milk could be used as an inexpensive and easily collectable matrix for SRLV detection. We therefore compared SRLV detection via two commercial enzyme-linked immunosorbent assays (ELISAs) and quantitative polymerase chain reaction (qPCR) in blood and corresponding milk samples from 321 goats originating from eight different SRLV-infected farms in Flanders (Belgium). The IDscreen® ELISA had a better relative sensitivity (97% vs 93%) and specificity (100% and 97%) than the Elitest® ELISA for SRLV-specific antibody detection in milk compared to serum. The higher sensitivity correlates with a 10-fold higher analytical sensitivity of the IDscreen® test. In contrast to the overall good ELISA results, qPCR on milk cell pellets lacked sensitivity (81%) and specificity (88%), compared to molecular detection in blood leucocyte pellets. Our results show that serology is more suitable than qPCR for SRLV diagnosis, and that milk may represent an interesting matrix for a preliminary evaluation of a herd’s infection status. Serum remains however the sample of choice for control programs where it is important to identify positive animals with the highest sensitivity.

scholarly journals Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

2014 ◽  
Vol 26 (6) ◽  
pp. 755-760 ◽  
Author(s):  
Maria J. Clavijo ◽  
Simone Oliveira ◽  
Jeffrey Zimmerman ◽  
Aaron Rendahl ◽  
Albert Rovira
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Systemic Disease ◽  
Analytical Sensitivity ◽  
Upper Respiratory Tract ◽  
Chain Reaction ◽  
Mycoplasma Hyorhinis ◽  
Field Samples ◽  
Polymerase Chain

Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

scholarly journals RT-qPCR Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

2021 ◽  
Author(s):  
Stephen Bustin ◽  
Sara Kirvell ◽  
Jim F Huggett ◽  
Tania Nolan
Keyword(s):  
Genomic Sequence ◽  
Public Awareness ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Sensitivity ◽  
Polymerase Gene ◽  
Chain Reaction ◽  
Rna Quantification ◽  
Viral Genes ◽  
Polymerase Chain ◽  
Rna Polymerase Gene

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target, since when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

scholarly journals Porcine reproductive and respiratory syndrome virus

2012 ◽  
Vol 24 (5) ◽  
pp. 855-866 ◽  
Author(s):  
Kerstin Wernike ◽  
Paolo Bonilauri ◽  
Malte Dauber ◽  
Jane Errington ◽  
Neil LeBlanc ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Negative Results ◽  
False Negative Results ◽  
Ring Trial ◽  
Polymerase Chain ◽  
Commercial Kits ◽  
The Individual

To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

Cerebral Ovine Herpesvirus 2 Infection of Cattle Is Associated With a Variable Neuropathological Phenotype

2020 ◽  
pp. 030098582097049
Author(s):  
Melanie M. Hierweger ◽  
Céline L. Boujon ◽  
Ronja V. Kauer ◽  
Mireille Meylan ◽  
Torsten Seuberlich ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Viral Encephalitis ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
The Central Nervous System ◽  
Consistent Feature ◽  
Polymerase Chain ◽  
The Brain

Cross-species infection with ovine herpesvirus 2 (OvHV-2) in cattle causes malignant catarrhal fever (MCF). MCF may involve the central nervous system (CNS) with necrotizing arteritis and/or vasculitis described to be unique to MCF and discriminatory compared to other viral CNS infections. However, a systematic histopathological characterization of the neural form of MCF in cattle is lacking. We examined medulla oblongata ( n = 9) or the entire brain ( n = 9) of 18 cattle in which OvHV-2 was identified by quantitative polymerase chain reaction (qPCR), in order to pinpoint potential variations in neuropathology. In 2/18 animals (11%) no lesions were identified, while 16/18 cattle (89%) had brain lesions of varying severity. Presence and quantities of OvHV-2 nucleic acid were determined by in situ hybridization and qPCR, respectively, and were related to the severity of lesions. Fifteen of 18 animals (83%) showed vasculitis, which was mainly of the lymphohistiocytic type, while pathognomonic necrotizing arteritis was only rarely present. Neuroparenchymal lesions included gliosis and/or neuronal changes in 7/16 brains with lesions (44%). The number of CD3+ lymphocytes was highest in animals with simultaneous vascular and neuroparenchymal lesions and high viral genome load. In one animal, OvHV-2 was exclusively observed in CD3+ lymphocytes but not in neurons or microglia. In conclusion, the neuropathological phenotype of bovine MCF in the brain was variable. In some cases, lesions mimicked neurotropic viral encephalitis, while pathognomonic necrotizing arteritis was not a consistent feature of neural MCF. Therefore, molecular detection of OvHV-2 is warranted in the presence of nonsuppurative encephalitis and in the absence of necrotizing arteritis.

scholarly journals Development of a novel magnetic particle-based agglutination immunoassay for anticardiolipin antibody detection in syphilis

2020 ◽  
Vol 96 (6) ◽  
pp. 411-416
Author(s):  
Mayur R Shukla ◽  
John W Deutsch ◽  
Lara E Pereira ◽  
Ellen N Kersh ◽  
Yetunde F Fakile
Keyword(s):  
Magnetic Particle ◽  
Treponema Pallidum ◽  
Anticardiolipin Antibody ◽  
Clinical Staging ◽  
Antibody Detection ◽  
Preliminary Evaluation ◽  
Analytical Sensitivity ◽  
The Novel ◽  
Serological Tests ◽  
Phosphate Buffered Saline

ObjectivesSerological tests of non-treponemal and treponemal types are the most frequently used for syphilis diagnosis. Treponemal tests are available in wide variety of assay formats; however, limited advances have been made for the improvement of conventional non-treponemal tests. The objective of this work was to develop a novel non-treponemal magnetic particle-based agglutination assay (NT-MAA) and evaluate its feasibility for syphilis testing.MethodsCardiolipin was modified and coupled to magnetic microbeads. Serum diluted in phosphate-buffered saline was mixed with cardiolipin-coupled beads and incubated in a round bottom microplate for 90–120 min followed by visual inspection. A panel of reported syphilis (n=127) and non-reactive (n=244) specimens was prepared to evaluate the NT-MAA performance in comparison to conventional rapid plasma reagin (RPR). Treponema pallidum particle agglutination (TP-PA) assay and enzyme immunoassay (EIA) were included. Analytical sensitivity and reproducibility of NT-MAA were also determined.ResultsThe non-treponemal NT-MAA and RPR showed sensitivity of 90.6% and 88.2% and specificity of 96.7% and 100%, respectively. The treponemal TP-PA and EIA yielded sensitivity of 100% and 99.2%, respectively, and 100% specificity by both assays. The per cent agreement between NT-MAA and RPR was 97% (kappa=0.931, 95% CI 0.891 to 0.971). Analytical sensitivity determined with IgM anticardiolipin antibody (ACA) was 2.6 µg/mL for both NT-MAA and RPR, while IgG ACA yielded 0.9 µg/mL and 1.7 µg/mL for NT-MAA and RPR, respectively. Qualitative results of intra-assay and interassay reproducibility revealed 100% consistency for NT-MAA.ConclusionPreliminary evaluation of the novel NT-MAA validated proof of concept using laboratory-characterised syphilis sera and demonstrated performance comparable to RPR. Further validation of NT-MAA using additional specimens with better clinical staging may broaden the scope of developed test for syphilis diagnosis.

scholarly journals MOLECULAR DETECTION OF ASTROVIRUS USING REVERSE TRANSCRIPTASE QUANTITATIVE POLYMERASE CHAIN REACTION TECHNIQUE

2020 ◽  
Vol 51 (Special) ◽  
Author(s):  
Fadhil & et al.
Keyword(s):  
Reverse Transcriptase ◽  
Molecular Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Melting Curve ◽  
Single Infection ◽  
Chain Reaction ◽  
Qpcr Analysis ◽  
Efficient Detection ◽  
Stool Samples ◽  
Polymerase Chain

This study was aimed to highlight the importance of the melt curve-quantitative reverse transcriptase PCR (RT-qPCR) analysis in the detection of astrovirus (AstV) from both negative and positive rotavirus and enterovirus (EVs) samples, and the effectiveness of the AstV infection on the vaccine immunogenicity in the vaccinated infected children.  By RT-qPCR based Sybre green associated- melting curve assay, stool samples of 49 enterovirus suspected patients and of 39 rotavirus suspected patients were tested for AstV. Results of EVs group showed a 29 (59.2%) positive AstV contributed to 26 (89.5%) co-infection with Evs and 3 (10.3%) as a single infection in negative samples for Evs. Furthermore, AstV co-infection percentage is higher than the single infection. Moreover, the percentage of the Astrovirus among the vaccinated AFP-suspected cases was 53%, while the percentage of these viruses among the unvaccinated was 100%. Thus, MamAstrovirus- 1 MK948878 is the first local isolate recorded in the Genbank. In conclusion, the RT-qPCR based on SYBR Green showed the rapid and efficient detection of AstV with few copies number. This allow to be used for the diagnosis of AstV along with other gastroenteritis viruses in a multiplex assay to reduce processing time.

scholarly journals Validation of a Triplex Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Traditional Protein Sources, Pisum sativum L. and Glycine max (L.) Merr., in Protein Powder Mixtures

2021 ◽  
Vol 12 ◽  
Author(s):  
Adam C. Faller ◽  
Dhivya Shanmughanandhan ◽  
Subramanyam Ragupathy ◽  
Yanjun Zhang ◽  
Zhengfei Lu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Glycine Max ◽  
Pisum Sativum ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Powder Mixtures ◽  
Protein Sources ◽  
Polymerase Chain

Several botanicals have been traditionally used as protein sources, including the leguminous Pisum sativum L. and Glycine max (L.) Merr. While a rich history exists of cultivating these plants for their whole, protein-rich grain, modern use as powdered supplements present a new challenge in material authentication. The absence of clear morphological identifiers of an intact plant and the existence of long, complex supply chains behoove industry to create quick, reliable analytical tools to identify the botanical source of a protein product (many of which contain multiple sources). The utility of molecular tools for plant-based protein powder authentication is gaining traction, but few validated tools exist. Multiplex quantitative polymerase chain reaction (qPCR) can provide an economical means by which sources can be identified and relative proportions quantified. We followed established guidelines for the design, optimization, and validation of qPCR assay, and developed a triplex qPCR assay that can amplify and quantify pea and soy DNA targets, normalized by a calibrator. The assay was evaluated for analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility. We tested the quantitative ability of the assay using pea and soy DNA mixtures, finding exceptional quantitative linearity for both targets – 0.9983 (p < 0.0001) for soy and 0.9915 (p < 0.0001) for pea. Ratios based on mass of protein powder were also tested, resulting in non-linear patterns in data that suggested the requirement of further sample preparation optimization or algorithmic correction. Variation in fragment size within different lots of commercial protein powder samples was also analyzed, revealing low SD among lots. Ultimately, this study demonstrated the utility of qPCR in the context of protein powder mixtures and highlighted key considerations to take into account for commercial implementation.

scholarly journals Rt-Qpcr Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

2021 ◽  
Author(s):  
Stephen Bustin ◽  
Sara Kirvell ◽  
Jim F Huggett ◽  
Tania Nolan
Keyword(s):  
Genomic Sequence ◽  
Public Awareness ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Sensitivity ◽  
Polymerase Gene ◽  
Chain Reaction ◽  
Rna Quantification ◽  
Viral Genes ◽  
Polymerase Chain ◽  
Rna Polymerase Gene

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target as when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

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