ABSTRACT
BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is one of the most important enteric viruses causing diarrhea in pigs. The establishment of a rapid detection method applicable in field conditions will be conducive to early detection of pathogen and implementation of relevant treatment. A novel nucleic acid amplification method, recombinase polymerase amplification (RPA), has been widely used for infectious disease diagnosis. RESULTS: In the present study, a reverse transcription (RT)-RPA assay combined with lateral flow dipstrip (LFD) was established for the visual detection of PEDV by targeting the N gene. The RT-RPA-LFD assay detected as low as 102 copies/µL of PEDV genomic RNA standard. Moreover, the novel RT-RPA-LFD assay did not show cross-reactivity with common swine pathogens, demonstrating high specificity. The performance of the assay for detection of clinical samples was also evaluated. A total number of 86 clinical samples were tested by RT-RPA-LFD and RT-PCR. The detection results of RT-RPA-LFD were compared with those of RT-PCR, with a coincidence rate of 96.5%. CONCLUSION: The newly established RT-RPA-LFD assay in our study had high sensitivity and specificity, with a potential to use in resource-limited areas and countries.
Subject(s)
Porcine epidemic diarrhea virus , Animals , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/veterinary , Porcine epidemic diarrhea virus/genetics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/veterinary , Recombinases/genetics , Reverse Transcription , Sensitivity and Specificity , SwineABSTRACT
The SARS-CoV-2 virus is the causative agent of COVID-19 and has undergone continuous mutations throughout the pandemic. The more transmissible Omicron variant has quickly spread and is replacing the Delta variant as the most prevalent strain globally, including in the United States. A new molecular assay that can detect and differentiate both the Delta and Omicron variants was developed. A collection of 660,035 SARS-CoV-2 full- or near-full genomes, including 169,454 Delta variant and 24,202 Omicron variant strains, were used for primer and probe designs. In silico data analysis predicted an assay coverage of >99% of all strains, including >99% of the Delta and >99% of Omicron strains. The Omicron variant differential test was designed based on the Δ31-33 aa deletion in the N-gene, which is present in the original B.1.1.529 main genotype, BA.1, as well as in BA.2 and BA.3 subtypes. Therefore, the assay should detect the majority of all Omicron variant strains. Standard curves generated with human clinical samples indicated that the PCR amplification efficiencies were 104%, 90.7% and 90.4% for the Omicron, Delta, and non-Delta/non-Omicron wild-type genotypes, respectively. Correlation coefficients of the standard curves were all >0.99. The detection limit of the assay was 14.3, 32.0, and 21.5 copies per PCR reaction for Omicron, Delta, and wild-type genotypes, respectively. The assay was designed to specifically detect SAR-CoV-2 strains. Selected samples with Omicron, Delta and wild-type genotypes identified by the RT-qPCR assay were also confirmed by sequencing. The assay did not detect any animal coronavirus-positive samples that were tested. Human nasal swab samples that previously tested positive (n = 182) or negative (n = 42) for SARS-CoV-2 by the ThermoFisher TaqPath COVID-19 Combo Kit, produced the same result with the new assay. Among positive samples, 55.5% (101/182), 23.1% (42/182), and 21.4% (39/182) were identified as Omicron, Delta, and non-Omicron/non-Delta wild-type genotypes, respectively.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , COVID-19/diagnosis , COVID-19/veterinary , Humans , Nucleic Acid Amplification Techniques/veterinary , RNA, Viral/genetics , SARS-CoV-2/geneticsABSTRACT
Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies µL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.
Subject(s)
Molecular Diagnostic Techniques/veterinary , Newcastle disease virus/genetics , Newcastle disease virus/isolation & purification , Nucleic Acid Amplification Techniques/veterinary , Animals , Columbidae , Feces/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral , Sensitivity and Specificity , Time FactorsABSTRACT
In this study, specific primers and fluorescent probes were designed to target the thymidine kinase (TK) gene sequence of avian infectious laryngotracheitis virus (ILTV). Through specificity and sensitivity tests, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method for detecting ILTV was established. The results showed that the method was specific and could be used to accurately detect ILTV, and there was no cross-reaction with Newcastle disease virus (NDV), avian influenza virus (AIV), or infectious bronchitis virus (IBV). Real-time fluorescence-based recombinase-aided amplification had high sensitivity, and the lowest detectable limit (LDL) for ILTV could reach 10 copies/µL, 1,000 times more sensitive than conventional PCR (104 copies/µL), to rival that of real-time fluorescence-based quantitative PCR (RFQ-PCR) (10 copies/µL). This method and RFQ-PCR were used to detect 96 samples of chicken throat swabs with ILT initially diagnosed in clinic from the north of China, and the coincidence rate of the 2 methods was 100%. The RF-RAA reaction required only 20-30 minutes to completing, and its sensitivity was much higher than that of conventional PCR. Real-time fluorescence-based recombinase-aided amplification is similar to RFQ-PCR and has the advantages of specificity, sensitivity, and high efficiency, so it is suitable for early clinical detection and epidemiological investigation of ILTV.