Development and validation of multiplex one-step qPCR/RT-qPCR assays for simultaneous detection of SARS-CoV-2 and pathogens associated with feline respiratory disease complex

PLoS ONE, Год журнала: 2024, Номер 19(3), С. e0297796 - e0297796

Опубликована: Март 22, 2024

Feline respiratory disease complex (FRDC) is caused by a wide range of viral and bacterial pathogens. Both Influenza A virus (IAV) Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) also induce diseases in cats. Two one-step multiplex qPCR/RT-qPCR assays were developed validated: FRA_1 (Feline assay 1) for the detection four targets FRA_2 three bacteria associated with FRDC. demonstrated high specificity, efficiency (93.51%–107.8%), linearity (> 0.998), analytical sensitivity (≤ 15 genome copies/μl), repeatability (coefficient variation [CV] < 5%), reproducibility (CV 6%). Among 63 clinical specimens collected from FRDC-suspected cats, 92.1% positive at least one pathogen co-infection was detected 57.1% samples. Mycoplasma felis (61.9%) most found pathogen, followed feline herpesvirus-1 (30.2%), Chlamydia (28.7%) calicivirus (27.0%). SARS-CoV-2 two specimens. In summary, this new panel constitutes useful reliable tool rapid pathogens FRDC

Язык: Английский

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One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus

Veterinary World, Год журнала: 2024, Номер unknown, С. 946 - 955

Опубликована: Май 1, 2024

Background and Aim: Feline coronavirus (FCoV), feline panleukopenia virus (FPV), leukemia (FeLV) are prevalent throughout China significantly threaten cat health. These viruses cause similar manifestations pathological damage. Rapid accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable rapid method for of FCoV, FPV, FeLV so that definite can be made effective measures taken prevent control viral infection. Materials Methods: We designed three pairs specific primers probes FCoV 5′ untranslated region, FPV protein 2, pol genes. Recombinant plasmid constructs were generated use as standard constructs. Optimal reaction conditions, including primer probe concentrations, cycles, annealing temperatures, obtained basis optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain (RT-qPCR) was successfully established simultaneously detect FeLV. The specificity, sensitivity, repeatability assay analyzed, its applicability validated by testing 1175 clinical samples. Results: RT-qPCR had high degree specificity only FeLV; it sensitivity with limits 139.904, 143.099, 152.079 copies/reaction p-FCoV, p-FPV, p-FeLV constructs, respectively, 0.06%–0.87% intra-assay coefficients variations. A total samples examined using RT-qPCR, positivity rates 18.47%, 19.91%, 47.57%, respectively. one-step 93.07% 97.99%, Conclusion: developed FeLV, which could used diagnostic tool monitoring diagnosis. Keywords: method, coronavirus, virus, multiplex reaction.

Язык: Английский

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Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

Viruses, Год журнала: 2023, Номер 15(9), С. 1881 - 1881

Опубликована: Сен. 5, 2023

Canine infectious respiratory disease complex (CIRDC) is the primary cause of in canine population and caused by a wide array viruses bacterial pathogens with coinfections being common. Since its recognition late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to dogs. Therefore, rapid detection differentiation SARS-CoV-2 from other common viral agents critical public health standpoint. Here, we developed validated panel four one-step multiplex qPCR/RT-qPCR assays for identification twelve associated CIRDC (canine adenovirus-2, distemper virus, herpesvirus-1, influenza A parainfluenza pneumovirus, coronavirus, SARS-CoV-2,

Язык: Английский

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A Quadruplex Reverse Transcription Quantitative Polymerase Chain Reaction for Detecting Canine Coronavirus, Canine Rotavirus, Canine Parvovirus, and Canine Distemper Virus

Microbiology Research, Год журнала: 2024, Номер 15(2), С. 746 - 761

Опубликована: Май 10, 2024

Background: Canine coronavirus (CCoV), canine rotavirus (CRV), parvovirus (CPV), and distemper virus (CDV) cause gastroenteritis in dogs, co-infections of these pathogens are common China. In particular, CCoV CRV confirmed to have important zoonotic potential public health issues. It is difficult diagnose diseases based only on clinical manifestations pathological damage. Methods: this study, four pairs specific primers probes targeting the M, VP7, CPV VP2, CDV N genes were designed. The reaction conditions, including primer probe concentrations, annealing temperatures, cycles, optimized for development a quadruplex RT-qPCR detection CCoV, CRV, CPV, CDV. assay was used test 1028 samples validate its application. Results: A successfully established differential CDV, with good specificity, high sensitivity, excellent repeatability. could specifically detect without cross-reactivity other viruses tested. showed sensitivity limits (LOD) 1.1 × 102 copies/reaction all plasmid constructs. repeatability, 0.05–0.90% intra-assay variation 0.02–0.94% inter-assay variation. tested using reported reference RT-qPCR. positivity rates 9.53%, 0.97%, 25.68%, 5.06% developed assay, 9.05%, 0.88%, 4.86% agreements higher than 99.32%. Conclusion: results indicated that rapid accurate differentiation

Язык: Английский

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Overview of Recent Advances in Canine Parvovirus Research: Current Status and Future Perspectives

Microorganisms, Год журнала: 2024, Номер 13(1), С. 47 - 47

Опубликована: Дек. 30, 2024

Canine parvovirus (CPV-2) was first identified in the late 1970s and has since become one of most significant infectious agents affecting dogs. CPV-2 causes severe diseases such as hemorrhagic gastroenteritis myocarditis, posing a major threat to canine health, particularly with high mortality rate puppies. It is globally recognized highly contagious lethal pathogen. CPV prone rapid mutation, leading emergence new variants. Despite widespread vaccination efforts, remains primary acute death young juvenile Furthermore, detection swine populations introduced additional challenges its control. This review summarizes current epidemiological status CPV, highlighting recent advancements diagnostic techniques vaccine development. Additionally, it discusses latest research on pathogenesis virus development antiviral agent proposes prevention control suggestions for under One Health concept. In particular, there need enhance surveillance viral dynamics, accelerate novel vaccines, deepen exploration underlying pathogenic mechanisms. aims provide scientific foundation effective guide future directions.

Язык: Английский

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Molecular Amplification and Cell Culturing Efficiency for Enteroviruses’ Detection in Cerebrospinal Fluids of Algerian Patients Suffering from Meningitis

Viruses, Год журнала: 2024, Номер 16(2), С. 170 - 170

Опубликована: Янв. 23, 2024

Enteroviruses (EVs) represent a major cause of viral meningitis, being responsible for nearly 1 billion infections each year worldwide. Several techniques were developed to obtain better diagnostic results EV infections. Herein, we evaluated the efficiency detection through isolation on both Rhabdomyosarcoma (RD) and Vero cell line cultures, conventional reverse transcription-polymerase chain reaction (RT-PCR) real-time RT-PCR. Thus, 50 cerebrospinal fluid (CSF) samples belonging patients suspected have meningitis in northern Algeria collected, anonymously numbered from subjected above-mentioned detection. Using RT-PCR, 34 CSF revealed be positive origin (68%). Thirteen them when RT-PCR was used (26%), only three gave culture technique (6%). Surprisingly, two culture-positive samples, namely, 31 39, negative using directly original samples. However, they turned amplification carried out their corresponding supernatant. The cell-cultured isolates then identified by sequencing genome’s VP1 regions. All belong echovirus 27 strain. This investigation demonstrates that are often more sensitive, accurate much faster, providing reliable within clinically acceptable timeframe. cultures remains crucial enough load serological tests or even avoid rare, but existing, false PCR.

Язык: Английский

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A multiplex RT-qPCR assay for simultaneous detection of cucurbit viruses from individual whitefly and plant samples

Plant Disease, Год журнала: 2024, Номер unknown

Опубликована: Июль 10, 2024

Whiteflies (Bemisia tabaci) are a significant pest of cucurbits and vector many viruses, leading to substantial economic losses. Modern diagnostic tools offer the potential for early detection viruses in whiteflies before crop production. One such tool is multiplex reverse transcriptase quantitative PCR (RT-qPCR) probe-based technique, which can detect multiple targets single reaction simultaneously quantify levels each target, with limit 100 copies per target. In this study, RT-qPCR–based system capable identifying one DNA virus three RNA whiteflies—cucurbit leaf crumple (CuLCrV), cucurbit chlorotic yellows (CCYV), yellow stunting disorder (CYSDV), squash vein yellowing (SqVYV)—was developed. To ensure reliability assay, an internal gene control as fifth target monitor false-negative results was incorporated. This newly developed molecular possesses several advantages. It up five desired from whitefly sample, even at concentrations low 1 ng/μl. evaluate its sensitivity, we conducted experiments using serially diluted cloned plasmids vitro transcribed transcripts viruses. We also assessed specificity assay by including aphid-transmitted other known infect cucurbits. The method successfully detected all allowed quantification mixture healthy RNA. Our aim study develop highly specific sensitive one-step RT-qPCR simultaneous transmitted offers advantages detection, enabling prompt measures mitigate further spread viral infections reduce yield Additionally, demonstrated ability mixed (CCYV, CYSDV, CuLCrV, SqVYV) individual number carried whitefly. outperforms currently available techniques detecting samples given time be effectively utilized monitoring plant symptomless plants.

Язык: Английский

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Development of a Quadruplex RT-qPCR for the Detection of Feline Kobuvirus, Feline Astrovirus, Feline Bufavirus, and Feline Rotavirus

Microbiology Research, Год журнала: 2024, Номер 15(4), С. 2129 - 2145

Опубликована: Окт. 21, 2024

Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), bufavirus (FeBuV), and rotavirus (FRV) are important pathogens for gastroenteritis, which is characterized by vomiting, diarrhea, dehydration. Four pairs of primers probes were designed to target the FeKoV VP1, FeAstV ORF2, FeBuV VP2, FRV NSP4 genes, a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay capable simultaneous detection four enteroviruses was developed after optimization reaction conditions. The established RT-qPCR showed high specificity, sensitivity, reproducibility. could detect discriminate FeKoV, FeAstV, FeBuV, FRV, but not other feline-related pathogens. limits (LODs) 109.761, 115.834, 125.481, 113.875 copies/reaction, respectively. intra- inter-assay coefficients variation (CV) 0.15–1.61% 0.15–1.59%, In all, 1869 clinical samples from Guangxi province in Southern China tested using assay, positivity rates 1.93%, 9.36%, 0.32%, 0.75%, These also reference assays, coincidence results between methods 99.63% 98.72% 100% (FRV), indicated that provide new method these viruses associated with gastroenteritis.

Язык: Английский

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Burden of Common Respiratory Pathogens Among Cats in China

Veterinary Medicine and Science, Год журнала: 2024, Номер 11(1)

Опубликована: Ноя. 22, 2024

ABSTRACT Background Feline respiratory disease complex (FRDC) is a set of illnesses which are primarily associated with different types viruses and bacteria. There scarcity data on pathogens FRDC in China. Objectives The primary objective this study was to investigate the prevalence dynamics Methods A total 458 samples were retrieved from veterinary clinics during 2021–2024 cats suffering infections. Four three bacteria targeted for molecular detection real time qPCR/RT‐qPCR assays. Results At least 1 pathogen detected 423 (92.3%), whereas no 7.7% samples. Bacteria 32.1% samples, 60.2% feline calicivirus (31.2%), herpesvirus‐1 (24.6%), influenza virus (2.8%) severe acute syndrome coronavirus‐2 (1.5%), rate Mycoplasma felis (15.5%), Chlamydia (10.2%) Bordetella bronchiseptica (6.3%). Significantly higher cases reported kittens (57.4%). Pathogen more common cold season. Mono‐infections involving one or 44.7% coinfections 47.5% No quadruple recorded study. Conclusions frequency alone combinations among diseased high, indicating heavy burden infections Kunshan, Continued surveillance desired, newly emerged should also be monitored routine diagnostic testing.

Язык: Английский

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Establishment of a Rapid Detection Method for Canine Coronavirus Using Recombinant Enzyme and Polymerase Mediated Isothermal Amplification Technology

Indian Journal of Microbiology, Год журнала: 2024, Номер unknown

Опубликована: Дек. 26, 2024

Язык: Английский

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