查看更多>>摘要：In this study, we combined reverse transcription recombinase polymerase amplification assay with the fluores-cence detection platform (qRT-RPA) and lateral flow biosensor (LFB RT-RPA) to allow for rapid detection of porcine encephalomyocarditis virus (EMCV). Primers and probes were designed to target the highly conserved region of 3D gene of porcine EMCV. The optimal reaction condition of qRT-RPA and LFB RT-RPA was set as 42 C for 20 min. The assays were highly specific to EMCV and no cross-reactions were observed with seven other porcine viruses. With a 10-fold serially diluted EMCV genomic RNA as template, the limit of detection was 1.0 x 10(2) and 1.0 x 10(1) copies for qRT-RPA assay and LFB RT-RPA assay, respectively. A total of 92 samples from different sources were examined using qRT-RPA, LFB RT-RPA and qRT-PCR. We found 100% diagnostic agreement between qRT-RPA (23/92) and qRT-PCR (23/92), and 97.83% diagnostic agreement between LFB RT-RPA (25/92) and qRT-PCR (23/92). There was no significant difference in performance between the RT-RPA assays developed in this study and a previously described qRT-PCR. However, RT-RPA assays were rapid and easy to perform while LFB RT-RPA exhibited higher sensitivity for EMCV than qRT-PCR. Therefore, the devel-oped EMCV RT-RPA assays provide an attractive and promising tool for effective detection of EMCV in low-resource settings.
El Bagoury, Gabr F.Elhabashy, RawanMahmoud, Ayman H.Hagag, Naglaa M....
9页
查看更多>>摘要：Foot-and-mouth disease (FMD) is an extremely contagious and economically important viral disease affecting livestock. Rapid and precise diagnosis of FMD is of critical importance for efficient control and surveillance strategies of the disease. In this study, one-step real-time reverse transcription-polymerase chain reaction (RTqPCR) assays were developed using newly designed primers/probe sets in the conserved regions within the VP1 coding sequence for specific detection of FMDV serotypes SAT 2 and O with their different lineage circulating in Egypt. The assays were validated for efficacy to detect different lineages of these endemic FMDV serotypes in Egypt; the detection limit was 10 genomic copies for serotype SAT 2 and one genomic copy for serotype O, with no cross-reactivity observed. These findings were confirmed by the specific and sensitive detection of FMDV in clinical samples obtained from different regions in Egypt and representing a range of subtypes within the SAT 2 and O serotypes. The results illustrated the potential of tailored RT-qPCR methods for the rapid detection and serotyping of FMDV belonging to different lineages of serotypes SAT 2 and O circulating in Egypt with high sensitivity and specificity. The developed assays could be easily deployed for routine surveillance and hence improving the disease control measures.
Gawande, S. P.Raghavendra, K. P.Monga, D.Nagrale, D. T....
11页
查看更多>>摘要：Cotton leaf curl disease (CLCuD) ranks top among all endemic diseases transmitted by whitefly (Bemisia tabaci) affecting cotton (Gossypium hirsutum) causing severe economic losses to the cotton growers in the Indian subcontinent. For its effective management, robust tools for detection are a prerequisite and it is important to diagnose the virus titre in early stage of infection in plants as well as in the disease transmitting vector. Considering the limitations in current PCR-based techniques we have standardised rapid and sensitive Loop Mediated Isothermal Amplification (LAMP) protocol for the diagnosis of cotton leaf curl virus (CLCuV) in cotton leaves and in its transmitting vector whitefly. Perhaps, this is the first report of use of LAMP tool for rapid diagnosis of CLCuV in cotton and its transmitting vector the whitefly. Further, the colorimetric detection for diagnostic simplicity of amplified LAMP product by using different dyes lead to enhanced applicability of this technique in the field of disease diagnostics. The merit of present study is that the diagnostic failure of PCR and LAMP due to low virus titre in the infected leaf has been circumvented through the combination of rolling circle amplification (RCA) with LAMP. Thus RCA-LAMP can be an option for ultra-sensitive detection of samples with low virus titre. The potential applications of this advanced diagnostic tool in laboratory research on diagnosis of CLCuV, an important viral pathogen of cotton have been discussed.
查看更多>>摘要：Metagenomic next-generation sequencing (mNGS) is a rapid deep-sequencing diagnostic tool for the unbiased identification of pathogens. In this study, we established a nanopore-sequencing-based mNGS protocol to detect two major viral pathogens of swine, Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine epidemic diarrhea virus (PEDV). Samples were spiked with the serially diluted viruses as standard references to define the specific protocols. The utility of the method was evaluated with key parameters. The limits of detection for PRRSV and PEDV were 2.3 x 10(2 )and 9.0 x 10(4) copies per reaction, respectively, and good correlations between PCR quantification cycle value and the mapped read count (log value) were observed. Only the nanopore reads could be assembled de novo into nearly full-length of the PRRSV genome, with 99.9% pairwise identity, and 90.0% genome coverage for PEDV. The established protocol was validated in PRRSV-positive clinical samples. The results for PRRSV-positive tissue and serum samples tested with mNGS protocol were 100% concordant with quantitative PCR results. The protocol also recognized infections of single or multiple viruses in a single sample. In conclusion, we have established a nanopore-sequencing-based mNGS protocol that efficiently identifies and characterizes viral pathogen(s) in a variety of clinical sample types.
查看更多>>摘要：The iron flocculation method, which comprises the Fe-virus flocculate formation-filtration-resuspension steps, is extensively used to concentrate and precipitate viruses distributed in water. To apply this method to concentrate white spot syndrome virus (WSSV) in seawater, viral genomic and infective recovery yields were compared between polyethylene sulfone (PES) and polycarbonate (PC) membrane filters and two types of resuspension buffers (oxalate and ascorbate). Viral genome quantitation was determined above a 95 % limit of detection (11.48 viral DNA copies/mu L) using quantitative real-time PCR. From WSSV-spiked seawater (100-106 viral DNA copies/mL), the viral genomic recovery yields of the PES-Oxalate, PC-Oxalate, PES-Ascorbate, and PC-Ascorbate conditions were 78.67 % +/- 12.90 %, 84.53 % +/- 24.30 %, 85.59 % +/- 16.98 %, and 93.74 % +/- 7.44 %, respectively. The detectable Fe-virus flocculates collected by the PC membrane were approximately 101 WSSV DNA copies/mL of seawater, a value more than 10-fold higher than that compared to the PES membrane filter (102 WSSV DNA copies/mL), regardless of the resuspension buffer types. WSSV resuspended with oxalate buffer caused mass mortality among whiteleg shrimp (Litopenaeus vannamei), inducing the expression of the virus en-velope protein, VP28, similar to that of a native virus, suggesting stable viral activity during the resuspension process. Based on the PES-Ascorbate, WSSV particles could be successfully concentrated in seawater from shrimp farms with white spot disease outbreaks (approximately 10(2) WSSV DNA copies/mL). Collectively, these findings indicate that the simple and efficient method of iron flocculation is sufficient to concentrate WSSV in seawater and could be used as a non-invasive approach and one of the reasonable diagnostic processes for white spot disease surveillance.
查看更多>>摘要：Goose astrovirus (GAstV) is a novel pathogen that was discovered in 2018. It has two genotypes, GAstV-1 and GAstV-2, and both can cause visceral gout of goslings and result in significant economic losses. The present work aimed to develop a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay to distinguish the two genotypes. MegAlign software was used to design two pairs of primers and a pair of matched probes based on the open reading frame 2 (ORF2) sequence with the greatest difference between GAstV-1 and GAstV-2, and primer and probe concentrations and annealing temperatures were optimised. Fluorescence signals were obtained for GAstV-1 and GAstV-2 in the FAM and VIC channels, respectively, but no fluorescent signal was observed for other pathogens. The detection limit for GAstV-1 and GAstV-2 was 33.3 and 33.7 DNA copies/mu L, respectively. Intra-and inter-assay variability tests revealed excellent reproducibility. Furthermore, the assay detected GAstV-1 and GAstV-2 in allantoic fluids (100% positive) spiked with viruses, and 70 clinical gout gosling samples were examined, of which 11.4% were positive for GAstV-1, 74.3% were positive for GAstV-2%, and 5.7% were positive for mixed infection. In summary, the developed duplex RT-qPCR assay has high specificity, sensitivity, and reproducibility, and can be used in the clinic for detection of GAstV-1 and GAstV-2.
Perelygina, Ludmila M.Chen, Min-HsinAbernathy, EmilyIcenogle, Joseph P....
7页
查看更多>>摘要：An examination of the nucleic acid sequence alignment of 48 full-length rubella virus genomes revealed that the 5' terminus of the genome is more conserved than the commonly used detection windows for rubella virus RNA located in the E1 protein coding region, suggesting that the 5' terminus could be a target for improving detection of all rubella virus genotypes. Two candidate primer sets were tested and the window between nucleotides (nts) 98 and 251 was found to have the greatest analytical sensitivity for detection of different genotypes. The new method had a limit of detection of four copies of rubella RNA per reaction with high specificity. The average coefficient variation of Ct was 2.2%. Concordance between the new method and currently used method, based on testing 251 clinical specimens collected from a rubella outbreak, was 99.4%. The assay was further improved upon by the incorporation of detection of both rubella virus RNA and mRNA from a cellular reference gene in a multiplex format. The multiplex format did not reduce the sensitivity or the reproducibility of rubella RNA detection and, of 60 specimens tested, the concordance between the single target and multiplex assays was 85.0%. To assess the utility of the multiplex assay for molecular surveillance, 62 rubella IgM positive serum samples from a rubella outbreak were tested, and eleven tested positive using the multiplex method while none were positive using the method targeting E1. These results show that the assay based on the new detection window near the 5' terminus of the genome can improve the detection of rubella virus for the purpose of molecular surveillance and case confirmation, with the added benefit of improved efficiency due to multiplexing.
Kim, Mi YoungKim, Hwa SuChung, Gyung TaePark, Younhee...
5页
查看更多>>摘要：Japanese encephalitis is prevalent throughout the temperate and tropical regions of Asia and is caused by the Japanese encephalitis virus (JEV), a mosquito-borne viral pathogen. The plaque reduction neutralization test (PRNT) is currently recommended as the gold standard test for detecting human antibodies against JEV. The plaque assay is the most widely used method for detecting infectious virions and involves counting discrete plaques in cells. However, it is time-consuming, and results can be subjective (owing to analyst variability during manual plaque counting). The focus reduction neutralization test (FRNT), which is based on an immunocolorimetric assay, can be used to automatically count foci formed by the JEV. Here, we compared the efficacy of PRNT and FRNT in measuring the neutralizing antibody titers using 102 serum samples from vaccinated and unvaccinated individuals. We observed positive correlations between these neutralization assays against the Nakayama and Beijing strains (R2 = 0.98 and 0.77, respectively). Thus, FRNT may be preferable to PRNT for evaluating the efficacy of JEV vaccines in large-scale serological studies.
查看更多>>摘要：Plum viroid I (PlVd-I) is found in marbling and corky flesh diseased plum trees in South Africa. In this study a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the high-throughput detection of PlVd-I was developed. This assay can be performed on crude extracts and detection can either be a pH dependent colorimetric reaction or a real-time fluorescent signal reaction. The false discovery rate was shown to be low and no decrease in sensitivity was detected compared to RT-PCR. The RT-LAMP assay allows for the fast and cost-effective detection of PlVd-I that will curtail the distribution of infected plant material.
NSTL
Elsevier