Isothermal Method of a Recombinase Polymerase Amplification Assay for the Detection of Most Common High-Risk Human Papillomavirus Type 16 and Type 18 DNA

Ma, Bach; Fang, Jian Jun; Wang, Ye; He, Haizhen; Dai, Mingyan; Lin, Wei Hsiang; Su, Wei; Zhang, Mingzhou

doi:10.7754/clin.lab.2016.160325

Cited by 17 publications

(19 citation statements)

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“…RPA-LF is thus much faster than qPCR and LAMP assays (1 h), Clinichip HPV TM (2.5 h), and ddPCR (>2 h). 33,68,69 Another unique advantage of RPA is its high sensitivity, capable of amplifying as few as 1-10 copies of target DNA. 70,71 Indeed, the LOD was 5-10 copies/reaction (Figure 4), similar to the HPV16/18 RPA assay described by Ma et al 33 The sensitivity is also comparable to qPCR and ddPCR.…”

Section: Discussionmentioning

confidence: 99%

“…33,68,69 Another unique advantage of RPA is its high sensitivity, capable of amplifying as few as 1-10 copies of target DNA. 70,71 Indeed, the LOD was 5-10 copies/reaction (Figure 4), similar to the HPV16/18 RPA assay described by Ma et al 33 The sensitivity is also comparable to qPCR and ddPCR. 72,73 As with ddPCR, we utilized PIK3CA as a reference gene to ensure that cfDNA was sufficient and amplifiable by the RPA reaction ( Figure 4).…”

Section: Discussionmentioning

confidence: 99%

“…24 RPA has been used for rapid detection of various pathogens, such as Escherichia coli O157:H7, 25 Orientia tsutsugamushi, 26,27 Rickettsia typhi, 27 Mycobacterium avium, 28 and HIV, 29,30 as well as for detection of cancer biomarkers, including TMPRSS2-ERG mRNA in urine for prostate cancer diagnosis, 31 EGFR mutations in lung cancer cells, 32 and HPV16/18 DNA for cervical cancer screening. 33 RPA can be combined with a lateral flow strip (RPA-LF) for rapid visualization by the naked eye while retaining sensitivity and being highly concordant with qPCR. 28,34 We hypothesized that HPV E7 cfDNA detection based on RPA-LF could provide rapid, simple, and point-of-care diagnostics of cervical cancer especially in developing countries.…”

Section: Impact Statementmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Impact Statementmentioning

confidence: 99%

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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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“…The exponential amplification is carried out on the target fragment by initiating a chain exchange reaction to form DNA synthesis [14]. The amplified products can typically be detected after operating only 5-10 min optimally at 37-40 • C. Since the first report in 2006, RPA has been emerging in medical diagnosis [15][16][17], foodborne pathogens [18,19], and genetically modified crops [20,21], as well as research on viruses [22,23]. For the detection of Salmonella, the fimY gene has been reported as unique to the Salmonella species and hence is suitable to use as an appropriate target gene for detecting Salmonella [24].…”

Section: Introductionmentioning

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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“…The replication is performed by a DNA polymerase, which has the strand-displacement activity necessary to synthesize a new complementary DNA strand [17]. The amplified products can be detected after being incubated at 37-42 • C in 5-20 min [18]. Accurate and simple readouts are important for the analysis of amplification products.…”

Section: Introductionmentioning

confidence: 99%

Multiplex Recombinase Polymerase Amplification Assay for the Simultaneous Detection of Three Foodborne Pathogens in Seafood

Chen

et al. 2020

Foods

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Foodborne pathogens can cause foodborne illness. In reality, one food sample may carry more than one pathogen. A rapid, sensitive, and multiple target method for bacteria detection is crucial in food safety. For the simultaneous detection of Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella Enteritidis, multi-objective recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) was developed in this study. The whole process, including amplification and reading, can be completed in 15 min at 37 °C. The detection limits were 2.6 × 101 CFU/mL for Staphylococcus aureus, 7.6 × 101 CFU/mL for Vibrio parahaemolyticus, and 1.29 × 101 CFU/mL for Salmonella Enteritidis. Moreover, colored signal intensities on test lines were measured by a test strip reader to achieve quantitative detection for Staphylococcus aureus (R2 = 0.9903), Vibrio parahaemolyticus (R2 = 0.9928), and Salmonella Enteritidis (R2 = 0.9945). In addition, the method demonstrated good recoveries (92.00%–107.95%) in the testing of spiked food samples. Therefore, the multiplex LFD-RPA assay is a feasible method for the rapid, sensitive, and quantitative detection of bacterial pathogens in seafood.

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Isothermal Method of a Recombinase Polymerase Amplification Assay for the Detection of Most Common High-Risk Human Papillomavirus Type 16 and Type 18 DNA

Abstract: Importantly, the RPA assay was demonstrated to be a useful and powerful method for detection of HPV virus, which therefore may serve as a valuable tool for rapid diagnosis of HPV infection in both commercial and clinical applications.

Cited by 17 publications

References 0 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

Multiplex Recombinase Polymerase Amplification Assay for the Simultaneous Detection of Three Foodborne Pathogens in Seafood

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