Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks

Hu, Jinqiang; Huang, Runna; Sun, Yuhua; Wei, Xiangke; Wang, Yi; Chun-peng, Jiang; Geng, Yangyang; Sun, Xiaoyan; Jianzhou, Jing; Gao, Hui; Wang, Zhangcun; Dong, Caiwen

doi:10.1016/j.mimet.2019.01.018

Cited by 52 publications

(27 citation statements)

References 37 publications

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“…75 Further optimization of an appropriate primer/probe combination is likely to improve assay specificity. 76 Regardless, our data revealed a satisfactory correlation (83.33-100%) between RPA-LF and ddPCR for HPV16 E7, HPV18 E7, and HPV16/18 E7. Hence, RPA-LF provides an alternative method to ddPCR for rapid and simple detection of HPV E7 cfDNA for cervical cancer monitoring.…”

Section: Discussionsupporting

confidence: 54%

Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker

Rungkamoltip

Temisak²,

Piboonprai

et al. 2020

Exp Biol Med (Maywood)

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Circulating cell-free DNA (cfDNA) has attracted attention as a non-invasive biomarker for diagnosing and monitoring various cancers. Given that human papillomavirus (HPV) DNA integration and overexpression of E6/E7 oncogenes are pivotal events for carcinogenesis, we sought to determine if HPV E7 cfDNA could serve as a specific biomarker for cervical cancer detection. We applied droplet digital PCR (ddPCR) to quantify HPV16/18 E7 cfDNA from the serum of patients with cervical cancer, cervical intraepithelial neoplasia, and controls. HPV16/18 E7 cfDNA was highly specific for cervical cancer, displaying 30.77% sensitivity, 100% specificity, and an area under the curve of 0.65. Furthermore, we developed a sensitive isothermal detection of HPV16/18 E7 and the PIK3CA WT reference gene based on recombinase polymerase amplification combined with a lateral flow strip (RPA-LF). The assay took less than 30 min and the detection limit was 5–10 copies. RPA-LF exhibited 100% sensitivity and 88.24% specificity towards HPV16/18 E7 cfDNA in clinical samples. The agreement between RPA-LF and ddPCR was 83.33% ( κ = 0.67) for HPV16 E7 and 100% ( κ = 1.0) for HPV18 E7, indicating a good correlation between both tests. Therefore, we conclude that HPV E7 cfDNA represents a potential tumor marker with excellent specificity and moderate sensitivity for minimally invasive cervical cancer monitoring. Moreover, the RPA-LF assay provides an affordable, rapid, and ultrasensitive tool for detecting HPV cfDNA in resource-limited settings.

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Section: Discussionsupporting

confidence: 54%

Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker

Rungkamoltip

Temisak²,

Piboonprai

et al. 2020

Exp Biol Med (Maywood)

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“…In general, the false-positive results were more likely to occur due to unexpected amplification or primer dimers [14]. An appropriate RPA primer/probe combination could avoid false positives or low sensitivity [29]. Furthermore, studies found that probes played an important role in RPA.…”

Section: Discussionmentioning

confidence: 99%

“…In the previous reports of literatures, LFD-RPA assay was applied to detect Salmonella in shellfish with a detection limit of 5 CFU/mL in 8 min at 40 • C [1]. The testing of Salmonella typhimurium in milk could successfully reach as low as 1.95 CFU/mL in 10 min at 40-42 • C [29]. Commercially available kits could obtain a reaction result within 15 min at 40 • C [34].…”

Section: Discussionmentioning

confidence: 99%

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

Fang

et al. 2019

Foods

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Salmonella can cause serious foodborne diseases. We have developed a lateral flow immunoassay combined with recombinase polymerase amplification (LFD-RPA) for detection of Salmonella in food. The conserved fragment (fimY) was selected as the target gene. Under an optimal condition (37 °C, 10 min), the sensitivity was 12 colony-forming units (CFU)/mL in a pure culture. Testing with 16 non-Salmonella strains as controls revealed that LFD-RPA was specific to the fimY gene of Salmonella. The established assay could detect Salmonella at concentrations as low as 1.29 × 102 CFU/mL in artificially contaminated samples. This detection was at a slightly higher level than that for a pure bacterial culture. Combined with the test strip reader, the LFD-RPA is a feasible method for quantitative detection of Salmonella based on the test line intensity, which was the ratio for the test line and control line of the reflected light. The method could be a potential point-of-care test in limited resource areas and provides a new approach and technical support for the diagnosis of food safety.

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“…Although PCR-based specific detection methods for V. alginolyticus targeting the toxR gene are promising, they cannot fulfill the current requirement for on-site detection by the mariculture industry because they require complicated thermal cycling devices and trained personnel [31]. Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technology that is fast, simple and specific [32].…”

Section: Introductionmentioning

confidence: 99%

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

Yü¹,

Zhao

Chen³

et al. 2020

BMC Vet Res

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Background: Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry because of its strong pathogenicity, quick onset after infection and high mortality rate in aquatic animals. Fast, simple and specific methods are needed for on-site detection to effectively control outbreaks and prevent economic losses. The detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR)-based molecular approaches have been developed, but their application is limited due to the requirement of complicated thermal cycling machines and trained personnel. Results: A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted the virulence gene toxR, which is reported to have good coverage for V. alginolyticus pathogenic strains. To ensure the specificity of the method, the primer-probe set of the RPA system was carefully designed to recognize regions in the toxR gene that diverge in different Vibrio species but are conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening of amplification performance, primer-dimer formation and false positive signals. The RPA-LFD method was confirmed to have high specificity for V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species or other pathogenic bacteria and was able to detect as little as 1 colony forming unit (CFU) per reaction without DNA purification, or 170 fg of genomic DNA, or 6.25 × 10 3 CFU/25 g in spiked shrimp without any enrichment. The method finishes detection within 30 min at temperatures between 35°C and 45°C, and the visual signal on the dipstick can be directly read by the naked eye. In an application simulation, randomly spiked shrimp homogenate samples were 100% accurately detected.

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Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks

Cited by 52 publications

References 37 publications

Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker

Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

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