sybr-green-i and Coronavirus-Infections

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID

Topics: Animals; Antibodies, Neutralizing; Bronchitis; Chickens; Coronavirus Infections; Infectious bronchitis virus; Poultry Diseases; Real-Time Polymerase Chain Reaction; Vaccines, Attenuated; Vaccines, Combined; Viral Vaccines

The duplex real-time PCR assay based on SYBR Green І was developed for detection of porcine epidemic diarrhea virus (PEDV) and porcine bocavirus (PBoV) 3/4/5 genotypes simultaneously. Two pairs of specific primers were designed targeting the N gene sequence of PEDV and VP1 gene sequence of PBoV3/4/5. PEDV and PBoV3/4/5 could be distinguished by their different melting temperatures (Tm) in one sample. The Tm value of PEDV was 83.5 °C, and the Tm value of PBoV3/4/5 was 78.5 °C, while other swine pathogens showed no specific melting peaks. The detection limits of this assay were 10 copies/μL for both PEDV and PBoV3/4/5. A total of sixty-three intestinal tissue samples were collected from piglets suffering from diarrhea, and the viral nucleic acids detected and identified by the real-time PCR assay and conventional PCR assay. The duplex real-time PCR detection results showed that the prevalence of PEDV and PBoV3/4/5 was 85.7% and 46%, respectively, and the co-infection rate of the two viruses was 28.6%. These results indicated that this duplex real-time PCR assay was a sensitive, specific and reproducible method for differentiating PEDV and PBoV3/4/5 or their co-infection.

Topics: Animals; Benzothiazoles; Bocavirus; Coinfection; Coronavirus Infections; Diamines; DNA Primers; Porcine epidemic diarrhea virus; Quinolines; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Swine; Transition Temperature

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 10

Topics: Adolescent; Adult; Animals; Benzothiazoles; Betacoronavirus; Child; Chlorocebus aethiops; Coronavirus Infections; COVID-19; Cross Reactions; Diamines; Fluorescent Dyes; Humans; Middle Aged; Nasopharynx; Organic Chemicals; Oropharynx; Pandemics; Pneumonia, Viral; Quinolines; Real-Time Polymerase Chain Reaction; RNA, Viral; SARS-CoV-2; Sensitivity and Specificity; Vero Cells; Young Adult

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus which was associated with severe diarrhea disease in pigs. SADS-CoV was first detected and identified as the causative agent of a devastating swine disease outbreak in southern China in 2017. Routine monitoring and early detection of the source of infection is therefore integral to the prevention and control of SADS-CoV infection. In this study, a SYBR green-based real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique was established for rapid detection and monitoring of this emerging virus. Specific primers were designed based on the conserved region within the M gene of the viral genome. The lowest detection limit of the RT-qPCR assay was 10 copies/μL. This assay was specific and had no cross-reaction with other 11 swine viruses. The positive rate of 84 clinical samples for the SYBR green-based RT-qPCR and the conventional RT-PCR was 73.81% (62/84) and 53.57% (45/84), respectively. These results demonstrated that the SYBR green-based RT-qPCR technique was an effectively diagnostic method with higher sensitivity than probe-based RT-qPCR and gel-based RT-PCR for detection and epidemiological investigations of SADS-CoV.

Topics: Alphacoronavirus; Animals; Benzothiazoles; China; Coronavirus Infections; Diamines; DNA Primers; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Staining and Labeling; Swine; Swine Diseases; Time Factors

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.

Topics: Animals; Benzothiazoles; Chickens; Coronavirus Infections; Diamines; Genotype; Infectious bronchitis virus; Lung; Nucleic Acid Denaturation; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Trachea

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED.

Topics: Animals; Benzothiazoles; Coronavirus Infections; Diamines; Organic Chemicals; Porcine epidemic diarrhea virus; Quinolines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Swine; Swine Diseases

A rapid, sensitive, and specific molecular method for the diagnosis of infectious bronchitis virus (IBV) infection is important in curbing infectious bronchitis outbreaks in Morocco and other countries.. In this study, an easy-to-perform SYBR green I real-time reverse transcriptase polymerase chain reaction (RT-PCR) targeting the nucleocapsid gene of IBV was developed and compared with conventional agarose gel-based RT-PCR for the detection of IBV infection.. We found that the SYBR green I real-time RT-PCR was at least 10 times more sensitive than the agarose gel electrophoresis detection method. The assay exhibited high specificity for IBV infection. All negative controls, such as Newcastle disease virus, infectious bursal disease virus, and avian influenza virus, were not detected.. The SYBR green I real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for diagnosis and control of infectious bronchitis outbreaks in Morocco. The test is a valuable and useful method as a routine assay for diagnosis of clinical IBV infection in commercial chickens.

Topics: Animals; Benzothiazoles; Bird Diseases; Chickens; Coronavirus Infections; Coronavirus Nucleocapsid Proteins; Diamines; Disease Outbreaks; Electrophoresis, Agar Gel; Infectious bronchitis virus; Morocco; Nucleocapsid Proteins; Organic Chemicals; Quinolines; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity