scholarly journals Diagnosis of feline haemoplasma infection using a real-time PCR assay

2004 ◽  
Vol 75 (2) ◽  
Author(s):  
R.G. Lobetti ◽  
S. Tasker
Keyword(s):  
Real Time ◽  
Biochemical Parameters ◽  
Inverse Correlation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Significant Inverse Correlation ◽  
Cytological Examination ◽  
Blood Smears ◽  
Polymerase Chain ◽  
Veterinary Laboratory

Haemobartonella felis has been reclassified within the genus Mycoplasma as Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum', collectively referred to as the feline haemoplasmas. A total of 78 cats from the Johannesburg area that had blood samples submitted to a private veterinary laboratory were tested using a real-time polymerase chain reaction (PCR) assay able to detect and distinguish the two feline haemoplasma (basonym Haemobartonella) species. All samples had been diagnosed with haemoplasma infection by cytological examination of blood smears. Statistical analysis was performed to evaluate associations between haemoplasma status, age, and haematological and biochemical parameters. On PCR assay 43 cats (55 %) were haemoplasma negative, 25 (32.1 %) positive for 'Candidatus Mycoplasma haemominutum', 5 (6.4 %) positive for Mycoplasma haemofelis and 5 (6.4 %) positive for both species. Significant inverse correlation was found between the amount of M. haemofelis DNA present in the blood and the haematocrit value. Cats that were positive for M. haemofelis showed macrocytic regenerative anaemia, monocytosis and thrombocytopaenia. This report documents the existence of both haemoplasma species in cats in South Africa.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

2021 ◽  
Author(s):  
Yang Pan ◽  
Jing Chen ◽  
Junhuang Wu ◽  
Yongxia Wang ◽  
Junwei Zou ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rapid Detection ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Canine Kobuvirus

Abstract Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

scholarly journals Novel genotype ofTritrichomonas foetusfrom cattle in Southern Africa

Parasitology ◽  
2016 ◽  
Vol 143 (14) ◽  
pp. 1954-1959 ◽  
Author(s):  
ANDREA CASTERIANO ◽  
UMBERTO MOLINI ◽  
KORNELIA KANDJUMBWA ◽  
SIEGFRIED KHAISEB ◽  
CAROLINE F. FREY ◽  
...  
Keyword(s):  
Real Time ◽  
Cysteine Proteases ◽  
Taqman Probe ◽  
Tritrichomonas Foetus ◽  
Chain Reaction ◽  
Pcr Assay ◽  
New Genotype ◽  
Reproductive Disease ◽  
Polymerase Chain ◽  
Novel Genotype

SUMMARYBovine trichomonosis caused byTritrichomonas foetusis a significant reproductive disease of cattle. Preputial samples were collected using sheath washing technique in bulls in Namibia. Thirty-six trichomonad cultures were characterized using the TaqMan-probe commercial real-time polymerase chain reaction (PCR) diagnostic assay (VetMAX™-Gold Trich Detection Kit) and CYBR real-time PCR assay based on TFR3/4 primers. Diagnostic real-time PCRs and DNA sequencing of the internal transcribed region confirmed presence ofT. foetusin 35 out of 36 samples. Multilocus genotyping using cysteine proteases (CP1, CP2, CP4, CP5, CP6, CP7, CP8, CP9) and malate dehydrogenase (MDH1) gene sequences demonstrate that theT. foetusin Namibia are genetically distinct from those characterized elsewhere. We report the discovery of a novel genotype ofT. foetusin Namibian cattle, distinct from otherT. foetusgenotypes in Europe, South and North America and Australia. We suggest recognition of a ‘Southern African’ genotype ofT. foetus. Identification of the new genotype ofT. foetusdemonstrates the need for wider global sampling to fully understand the diversity and origin ofT. foetuscausing disease in cattle or cats.

scholarly journals Collaborative Trial for Validation of a Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay for Detection of Central Nervous System Tissues as Bovine Spongiform Encephalopathy RiskMaterial: Part 1

2006 ◽  
Vol 89 (5) ◽  
pp. 1335-1340
Author(s):  
Amir Abdulmawjood ◽  
Holger Schnenbrcher ◽  
Michael BÜlte
Keyword(s):  
Central Nervous System ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Products ◽  
Spongiform Encephalopathy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Heat Treated ◽  
Polymerase Chain

Abstract A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120C) and the medium heat-treated samples (sausages cooked at 80C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

Detection and Quantitation of Enterohemorrhagic Escherichia coli O157, O111, and O26 in Beef and Bovine Feces by Real-Time Polymerase Chain Reaction†

2002 ◽  
Vol 65 (9) ◽  
pp. 1371-1380 ◽  
Author(s):  
VIJAY K. SHARMA
Keyword(s):  
Real Time ◽  
Shiga Toxin ◽  
Enterohemorrhagic Escherichia Coli ◽  
Specific Primer ◽  
Chain Reaction ◽  
Pcr Assay ◽  
E Coli ◽  
Taqman Probes ◽  
Polymerase Chain ◽  
Pcr Assays

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and certain non-O157 EHEC serotypes (such as O26:H11, O26: NM, O111:H8, and O111:NM) have emerged as significant causes of human disease throughout the world. Important virulence attributes of EHEC are the intimin protein (encoded by the eae gene) and Shiga toxins 1 and 2 (encoded by the stx1 and stx2 genes, respectively). Two sets of real-time polymerase chain reaction (R-PCR) assays were developed for the simultaneous detection and quantitation of EHEC through the monitoring of the presence of the eae and stx genes, and these assays were evaluated. In the eaeR-PCR assay, three sets of primers and TaqMan probes were designed for the amplification and real-time detection of a portion of the eae gene specific to the EHEC O26, O111, and O157 serotypes. In the stxR-PCR assay, two sets of primers and TaqMan probes were used to amplify and detect the stx1 and stx2 genes. DNA prepared from 67 bacterial strains carrying known virulence markers was tested to determine the specificities of the two assays. In the eaeR-PCR assay, eaeO157- and eaeO111-specific primer-probe sets identified only EHEC O157 and O111 strains, respectively. The eaeO26-specific primer-probe set identified all EHEC O26 isolates and some Shiga toxin–negative serotypes of enteropathogenic E. coli and rabbit diarrheagenic E. coli. The stxR-PCR assay was able to identify only those strains carrying either or both of the Shiga toxin–encoding genes. The detection range of both R-PCR assays was linear over DNA concentrations corresponding to 103 to 108 CFU/ml of an EHEC strain. Both assays were able to detect and quantify very low levels (1 to 10 CFU/g of food or feces) of EHEC in feces and ground beef enriched for 16 h in a modified Trypticase soy broth. In conclusion, eae- and stx-based R-PCR assays are reliable and sensitive methods for the rapid screening and specific and quantitative detection of important serotypes of EHEC in cattle and in foods of bovine origin.

Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea

2004 ◽  
Vol 72 (3) ◽  
pp. 496-501 ◽  
Author(s):  
Xiaoli L. Pang ◽  
Bonita Lee ◽  
Nasim Boroumand ◽  
Barbara Leblanc ◽  
Jutta K. Preiksaitis ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Stool Specimens ◽  
Polymerase Chain

scholarly journals Evaluation of Amplification Targets for the Specific Detection ofBordetella pertussisUsing Real-Time Polymerase Chain Reaction

2014 ◽  
Vol 25 (4) ◽  
pp. 217-221 ◽  
Author(s):  
Mohammad Rubayet Hasan ◽  
Rusung Tan ◽  
Ghada N Al-Rawahi ◽  
Eva Thomas ◽  
Peter Tilley
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Target Genes ◽  
Reference Panel ◽  
Superior Performance ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

BACKGROUND:Bordetella pertussisinfections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detectB pertussisare typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.METHODS: A novelB pertussisreal-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481,ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including differentBordetellaspecies and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products.RESULTS: Analytical sensitivity was highest for the assay targeting the IS481element; however, the assay lacked specificity forB pertussisin the reference panel and in the clinical samples. False-positive results were also observed with assays targeting theptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains ofB pertussis. However, a novel assay targeting the porin gene demonstrated high specificity forB pertussisboth in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted allB pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction.CONCLUSION: A novel porin assay forB pertussisdemonstrated superior performance and may be useful for improved molecular detection ofB pertussisin clinical specimens.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

scholarly journals Systematic Review and Meta-analysis of Real-time Polymerase Chain Reaction assay for the detection of COVID-19 from clinical samples in low-and middle-income countries: Protocol

2021 ◽  
Author(s):  
Emmanuel Oladipo Babafemi
Keyword(s):  
Systematic Review ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meta Analysis ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Middle Income ◽  
Polymerase Chain

Abstract Background: COVID-19 has spread globally since its discovery in Hubei province, China in December 2019 and became pandemic in 2020. COVID-19 is a new betacoronavirus and a variant of severe acute respiratory syndrome coronavirus 2 (SARA- CoV-2). Rapid, accurate and reliable diagnosis of COVID-19 will prevent the spread and allow for appropriate management. The main objective of this systematic review is to identify, appraise and summarise the published evidence on the diagnostic performance and effectiveness of SARS-CoV-2 virus in the diagnosis of current or previous COVID-19 using real-time polymerase chain reaction (RT-PCR) assay in low-and middle-income countries (LMICs). Methods: We will search MEDLINE/PubMed, EMBASE, BIOSIS, LILACS, Cochrane Infectious Diseases Group Specialised Register (CIDG SR), Global Health, and CINAHL for published studies for the diagnosis of COVID-19 using real-time polymerase chain reaction assay in LMICs There will be no restriction regarding the language, date of publication, and publication status. We will include retrospective, cross-sectional and cohort observational studies will be included in the review. Selection of studies, data extraction and management, assessment of risk of bias, and quality of evidence will be performed by two independent reviewers (EB and BC). A third researcher (GM) will be consulted in case of discrepancies. Depending on the availability and quality of the data, a meta-analysis will be performed. Otherwise, findings will be qualitatively reported. Discussion: To our knowledge, this is the first systematic review and meta-analysis to assess the uptake of RT-PCR assay for SARS-CoV-2 detection from clinical samples in human in LMICs. This review will make available evidence on the uptake, accuracy, approach, and interpretation of results of this assay in the context of COVID-19 diagnosis which will meet an urgent need, considering the diagnostic challenges of RT-PCR assay for COVID-19 diagnosis in humans. Systematic review registration: PROSPERO CRD42021271894

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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