Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2

Liu, Libing; Wang, Jianchang; Geng, Yunyun; Wang, Jinfeng; Li, Ruiwen; Shi, Ruihan; Yuan, Wanzhe

doi:10.1016/j.mcp.2018.04.004

Cited by 19 publications

(13 citation statements)

References 32 publications

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“…The RPA reaction opens the two strands of the double-stranded DNA by the enzyme and amplifies the DNA target with the strand-displacing activity isothermally. DNA targets are exponentially amplified for detection within 20 min in a temperature range of 37-42 • C (Liu et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer–Dimers

Wang¹,

Zhao

Si³

et al. 2020

Front. Microbiol.

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Listeria monocytogenes is an important foodborne pathogenic bacterium that is explicitly threatening public health and food safety. Rapid, simple, and sensitive detection methods for this pathogen are of urgent need for the increasing on-site testing demands. Application of the isothermal recombinase polymerase amplification (RPA) and the lateral flow strip (LFS) in the detection is promising for fast speed, high sensitivity, and little dependency on equipment and trained personnel. However, the simplicity comes with an intrinsic and non-negligible risk, the false-positive signals from primer-dimers. In this study, an improved RPA-LFS system was established for detection of L. monocytogenes that eliminated false-positive signals from primerdimers. Primer candidates were carefully selected from the entire L. monocytogenes genome sequence and rigorously screened for specific amplifications in PCR and RPA reactions. For the optimal primer pairs, probes that matched the targeted fragment sequences, although had the smallest chance to form cross-dimers with the primers, were designed and screened. The intelligent use of the probe successfully linked the positive signal to the actual amplification product. This RPA-LFS system was highly specific to L. monocytogenes and was able to detect as low as 1 colony-forming unit of the bacterium per reaction (50 µl) without DNA purification, or 100 fg of the genomic DNA/50 µl. The amplification could be conducted under the temperature between 37 and 42 • C, and the whole detection finished within 25 min. Test of artificially contaminated milk gave 100% accuracy of detection without purification of the samples. Various food samples spiked with 10 colony-forming unit of L. monocytogenes per 25 g or 25 ml were successfully detected after an enrichment time period of 6 h. The RPA-LFS system established in this study is a rapid, simple, and specific detection method for L. monocytogenes that has eliminated false-positive results from primer-dimers. In

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Section: Introductionmentioning

confidence: 99%

Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer–Dimers

Wang¹,

Zhao

Si³

et al. 2020

Front. Microbiol.

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“…The sensitivity of PCR-LFIA with standard DNA in this study was 3 × 10 1 copies/μL. The sensitivity was also higher than conventional PCR and recombinase polymerase amplification assay [41, 42]. The verification result of PCR-LFIA showed a similar sensitivity to the previously reported qPCR and real-time recombinase polymerase amplification assay [15, 43], while the inexpensive equipment required for PCR-LFIA makes the latest valuable molecular test method, especially for resource limited setting.…”

Section: Discussionmentioning

confidence: 76%

Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2

Zhuang

Ji²,

Tian

et al. 2019

BMC Vet Res

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BackgroundCanine parvovirus 2 (CPV-2) is one of the most common etiological agents that cause severe gastroenteritis in puppies. Early accurate diagnosis is important for infected dogs. In recent years, magnetic separation has become an efficient and useful tool for bioassays. In this study, polymerase chain reaction (PCR) combined with fluorescent lateral flow immunoassay (LFIA) based on magnetic purification assay was developed for the quantitative detection of CPV-2.ResultsThe optimum working reaction volume and reaction time for LFIA was 100 μL and 2 min, respectively. The PCR-LFIA assay only detected CPV-2, and did not show cross-detection of non-CPV strains. Experiments showed analytical sensitivity of 3 × 101 copies/μL and demonstrated the PCR-LFIA has a diagnostic agreement of 100% with conventional PCR on detection of clinical samples (22.6% positive, 14/62). Cutoff value is 146. The results were further verified by sequencing and BLAST software. The entire process from PCR step only takes ~ 80 min.ConclusionsThis approach provides an attractive platform for rapid and quantitative detection of CPV-2, indicating great promise as a convenient molecular detection tool to facilitate disease outbreak investigations and response timely.

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“…The amplification is first-performed for 15 min followed by visualization of results with naked eye on the LFS within 5 min. The assay could detect CPV-2a, CPV-2b, and CPV-2c with a detection limit of 1.0 × 10 2 copies per reaction, which was the same as that of a real-time PCR (Liu et al, 2018). Recently, a cost effective paper-based cell-free transcription–translation reactions for Norovirus has been devised.…”

Section: Enteric Virus Detection Methodsmentioning

confidence: 76%

Advances in Diagnostic Approaches for Viral Etiologies of Diarrhea: From the Lab to the Field

et al. 2019

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The applications of correct diagnostic approaches play a decisive role in timely containment of infectious diseases spread and mitigation of public health risks. Nevertheless, there is a need to update the diagnostics regularly to capture the new, emergent, and highly divergent viruses. Acute gastroenteritis of viral origin has been identified as a significant cause of mortality across the globe, with the more serious consequences seen at the extremes of age groups (young and elderly) and immune-compromised individuals. Therefore, significant advancements and efforts have been put in the development of enteric virus diagnostics to meet the WHO ASSURED criteria as a benchmark over the years. The Enzyme-Linked Immunosorbent (ELISA) and Polymerase Chain Reaction (PCR) are the basic assays that provided the platform for development of several efficient diagnostics such as real-time RT-PCR, loop-mediated isothermal amplification (LAMP), polymerase spiral reaction (PSR), biosensors, microarrays and next generation sequencing. Herein, we describe and discuss the applications of these advanced technologies in context to enteric virus detection by delineating their features, advantages and limitations.

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Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2

Cited by 19 publications

References 32 publications

Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer–Dimers

Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer–Dimers

Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2

Advances in Diagnostic Approaches for Viral Etiologies of Diarrhea: From the Lab to the Field

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