scholarly journals Development of a Recombinase Polymerase Amplification Fluorescence Assay for the Detection of Canine Adenovirus 2

2021 ◽  
Vol 8 ◽  
Author(s):  
Li Xiao ◽  
Mengdi Zhang ◽  
Zhige Tian ◽  
Ye Ge ◽  
Tongyuan Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Canine Distemper Virus ◽  
Pathogen Detection ◽  
Detection Method ◽  
Adenovirus Type ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Clinical Applicability ◽  
Field Samples

Canine adenovirus type 2 (CAdV-2) is often found in co-infections with other pathogens causing canine infectious respiratory disease (CIRD). Rapid, efficient, and convenient pathogen detection is the best approach for early confirmatory diagnosis. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) assay for detection of canine adenovirus 2 (CAV), which can detect CAV within 15 min at 39°C. The detection limit that assay was 214 copies/μl DNA molecules per reaction. The specificity was indicated by a lack of cross-reaction with canine distemper virus (CDV), canine coronavirus (CCV), and canine parvovirus (CPV). Field and clinical applicability of this assay were evaluated using 86 field samples. The coincidence rate of the detection results for clinical samples between CAV-RPA and qPCR was 97.7%. In summary, the real-time CAV-RPA analysis provides an efficient, rapid and sensitive detection method for CAV.

scholarly journals Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification

2021 ◽  
Vol 12 ◽  
Author(s):  
Bailin An ◽  
Hongbin Zhang ◽  
Xuan Su ◽  
Yue Guo ◽  
Tao Wu ◽  
...  
Keyword(s):  
Real Time ◽  
Detection Method ◽  
Reaction System ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Specific Detection ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Polymerase Chain ◽  
The One

Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 102 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (100 copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.

scholarly journals Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit

Critical Care ◽  
2010 ◽  
Vol 14 (4) ◽  
pp. R159 ◽  
Author(s):  
Katsunori Yanagihara ◽  
Yuko Kitagawa ◽  
Masao Tomonaga ◽  
Kunihiro Tsukasaki ◽  
Shigeru Kohno ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Dna Detection ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Pathogen Dna

scholarly journals Development of an Efficient Real-Time Quantitative PCR Protocol for Detection ofXanthomonas arboricolapv. pruni inPrunusSpecies

2010 ◽  
Vol 77 (1) ◽  
pp. 89-97 ◽  
Author(s):  
Ana Palacio-Bielsa ◽  
Jaime Cubero ◽  
Miguel A. Cambra ◽  
Raquel Collados ◽  
Isabel M. Berruete ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Plant Material ◽  
Pathogen Detection ◽  
Plant Protection ◽  
The European Union ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Mediterranean Plant ◽  
Different Origins

ABSTRACTXanthomonas arboricolapv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as beingX. arboricolapv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system inX. arboricolapv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 102CFU ml−1, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed forX. arboricolapv. pruni strains from different origins as well as for closely relatedXanthomonasspecies, non-Xanthomonasspecies, saprophytic bacteria, and healthyPrunussamples. The efficiency of the developed protocol was evaluated with field samples of 14Prunusspecies and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, andX. arboricolapv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test forX. arboricolapv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

scholarly journals Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification: Testing for Specificity Using a Standardized Library of Isolates

2009 ◽  
Vol 99 (4) ◽  
pp. 390-403 ◽  
Author(s):  
F. N. Martin ◽  
M. D. Coffey ◽  
K. Zeller ◽  
R. C. Hamelin ◽  
P. Tooley ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Real Time ◽  
Pathogen Detection ◽  
Species Group ◽  
False Positives ◽  
Phytophthora Ramorum ◽  
Sscp Analysis ◽  
Oxidase Gene ◽  
Phytophthora Spp ◽  
Field Samples

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as β-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

scholarly journals Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246573
Author(s):  
Sandeep K. Gupta ◽  
Qing Deng ◽  
Tanushree B. Gupta ◽  
Paul Maclean ◽  
Joerg Jores ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
The Real ◽  
Positive Rate ◽  
Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

scholarly journals Development and Evaluation of Real-Time Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of West Nile Virus in Human Clinical Samples

2021 ◽  
Vol 10 ◽  
Author(s):  
Priyanka Singh Tomar ◽  
Sanjay Kumar ◽  
Sapan Patel ◽  
Jyoti S. Kumar
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Reverse Transcription ◽  
Febrile Illness ◽  
Posterior Uveitis ◽  
Qpcr Assay ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
West Nile Fever ◽  
West Nile

West Nile virus (WNV) causes West Nile fever and encephalitis worldwide. Currently, there are no effective drugs or vaccines available in the market to treat WNV infection in humans. Hence, it is of paramount importance to detect WNV early for the success of the disease control programs and timely clinical management in endemic areas. In the present paper, we report the development of real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for rapid and real-time detection of WNV targeting the envelope (env) gene of the virus. The RPA reaction was performed successfully at 39°C for 15 min in a real-time thermal cycler. The sensitivity of this assay was found similar to that of the quantitative real-time RT PCR (RT-qPCR) assay, which could detect 10 copies of the gene. The efficacy of the assay was evaluated with a panel of 110 WN suspected human samples showing the signs of retinitis, febrile illness and acute posterior uveitis. In comparison with RT-qPCR, RT-RPA showed a specificity of 100% (CI, 95.07–100%) and sensitivity of 96.15% (CI, 80.36–99.90%) with a negative (NPV) and positive predictive value (PPV) of 98.65 and 100%, respectively. The level of agreement between RT-RPA and reference RT-qPCR assay was shown to be very high. The turnaround time of real-time RPA assay is about 10-20 times faster than the RT-qPCR, which confirms its utility in the rapid and sensitive diagnosis of WNV infection. To the best of our knowledge, this is the first report which deals with the development of real-time RT-RPA assay for simple, rapid, sensitive, and specific detection of WNV in human clinical samples. The present RT-RPA assay proves to be a powerful tool that can be used for the rapid diagnosis of a large number of patient samples in endemic settings.

scholarly journals 657. Evaluation of Nanopore-Based 16S Ribosomal RNA (rRNA) Gene Sequencing for the Development of a Rapid Infection Intervention Clinical Service

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S302-S302
Author(s):  
Rahul Batra ◽  
R Baldan ◽  
P Cliff ◽  
Amita Patel ◽  
Jonathan Edgeworth ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Pathogen Detection ◽  
Clinical Service ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Acid Extraction ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Gene Assay

Abstract Background Rapid and accurate identification of bacteria is the basis of appropriate antibiotic treatment and effective clinical decision-making. Next-generation sequencing (NGS) platforms such as Oxford Nanopore Technologies (ONT) holds the promise of a diagnostic revolution by overcoming the limitations of culture-based identification with rapid molecular detection of bacteria. We have developed a pilot to evaluate an ONT 16S rRNA gene assay with the ability to provide real-time analysis and identification of bacterial species. Our aim was to investigate whether long-read sequencing and high-speed analysis can be combined to create a clinically useful, rapid diagnostic tool. Methods A collection of bacterial isolates representing pathogenic species received by the clinical laboratory over 1 year was assembled. Sample preparation was as described in the ONT 16S protocol and included bead beating sample disruption, MagNA Pure automated nucleic acid extraction (Roche), and PCR amplification (Thermo). Sequencing was performed on the MinION and GridION X5 platforms. Output was analyzed with ONT’s automated EPI2ME 16S pipeline which assigns reads to taxa using BLAST results and the NCBI 16S Bacterial database. Results A total of 155 clinical samples with 139 species were sequenced. 119 species were identified at the species level. For 20 samples, a species in the same genus claimed the majority of reads, with the true species being matched to 3%-41% of reads. The average proportion of reads assigned to the correct species was 62.2%, specifically 67% for non-Enterobacteriaceae and 33% for Enterobacteriaceae. 4 clinical samples (3 Bronchoalveolar lavages (BALs), positive for (1) K. pneumoniae, (2) S. pneumoniae, and (3) S. pneumoniae, S. enterica, and S. typhimurium, and 1 bone positive for P. aeruginosa) were also analyzed with sequencing results matching culture. Conclusion Early results show that 16S rRNA sequencing coupled with real-time analysis was able to accelerate pathogen detection and was able to discriminate the majority of species from a relevant clinical collection. Pipeline refinement is required and subsequent confirmatory consensus-based identification may be a helpful adjunct. Nanopore sequencing shows promise as a rapid bacterial pathogen detection platform for clinical service. Disclosures All authors: No reported disclosures.

scholarly journals Development of recombinase polymerase amplification assays for rapid and visual detection of canine distemper virus infecting giant panda

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Pei Huang ◽  
Yue Yu ◽  
Xianyong Meng ◽  
Tiecheng Wang ◽  
Feihu Yan ◽  
...  
Keyword(s):  
Canine Distemper Virus ◽  
Giant Panda ◽  
Limit Of Detection ◽  
Natural Infection ◽  
Rapid Test ◽  
Recombinase Polymerase Amplification ◽  
N Gene ◽  
Clinical Samples ◽  
Canine Distemper ◽  
Giant Pandas

Abstract Background Canine distemper virus (CDV) is an enveloped negative-strand RNA virus that exhibits a high mutation rate and continuously expands the range of hosts. Notably, CDV has infected giant panda with spill over from viral reservoirs in canines. Giant pandas (Ailuropoda melanoleuca), especially captive pandas, are known to be susceptible to natural infection with CDV. The high fatality rate of CDV poses a serious threat to the safety of the giant panda population. However, vaccines or drugs for canine distemper in giant pandas have not been developed to date. Therefore, a rapid test that can achieve accurate onsite detection of CDV is important to enable the timely implementation of control measures. In this study, we established a nucleic acid visualization assay for targeting the CDV N gene by using combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). Results The RT-RPA-VF assay does not require sophisticated equipment, and it was determined to provide rapid detection at 35 °C for 30 min, while the limit of detection was 5 × 101 copies/μl RNA transcripts and 100.5 TCID50 ml− 1 viruses. The results showed that the assay was high specific to CDV and had no cross-reactivity with other viruses infecting the giant panda. Compared with RT-qPCR, RT-RPA-VF assay had a sensitivity of 100% and a specificity of 100% in 29 clinical samples. The coincidence rate between RT-RPA-VF and RT-qPCR was 100% (kappa = 1), indicating that the RT-RPA-VF assay possessed good diagnostic performance on clinical samples. Conclusions The RT-RPA-VF provides a novel alternative for the simple, sensitive, and specific identification of CDV and showed great potential for point of care diagnostics for captive and wild giant panda.

scholarly journals Prevalence of causative agents of respiratory infections in cats and dogs in Russia

2020 ◽  
Vol 2020 (1) ◽  
pp. 9-13
Author(s):  
Sergey Konyaev
Keyword(s):  
Real Time ◽  
Respiratory Infections ◽  
Adenovirus Type ◽  
Bordetella Bronchiseptica ◽  
Feline Calicivirus ◽  
Infectious Agents ◽  
Reverse Transcription Pcr ◽  
Parainfluenza Viruses ◽  
Causative Agents

Feline calicivirus (FCV), as well as the bacteria Bordetella bronchiseptica, Chlamydia felis and Mycoplasma felis are the top five most common pathogens that cause URTI. A Kennel Cough or Infectious Respiratory Disease in Dogs (IRS) has a complex etiological cause. The purpose of this study is to analyze the prevalence of the main pathogens that cause URTI and IRS entering the veterinary clinics of Russia. By real time PCR (profile) from material obtained from 5 520 cats for 5 infectious agents, 81.7 % were positively detected, and in 25.9 % of cases with the appearance of co-infections. The DNA of the bacterium B. bronchiseptica was detected 3.7 % (n = 233), C. felis 11.2 % (n = 698), M. felis 21.5 % (n = 1347). FCV RNA was detected in real time reverse transcription PCR in 21.4 % of cases (n = 1340), FHV-1 DNA in 16.3 % (n = 1017) of samples. When examining 1742 dogs for six causative agents of the IRRS: parainfluenza viruses (CPiV), distemper (CDV), adenovirus type 2 (CAV-2), canine herpesvirus (CHV), B. bronchiseptica, Mycoplasma cynos ― positive 981 were detected samples (56.3 %), the only pathogen was detected in 740 cases (75.4 %). In 206 cases, B.bronchiseptica DNA was detected, in 13 CAV-2, in 272 CPiV, and in 84 CDV RNA, CHV ― 17, M.cynos ― 389. Thus, the proportion of positive reactions arriving at B. bronchiseptica was 15.9% , for CAV-2 ― 1.01 %, for cases of CPiV ― 20.8 %, and for CDV ― 6.4 %, for M. cynos29.8 %, CHV 1.3 %, for mix infection ― 24.6 %.

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