Development of a novel genotype-specific loop-mediated isothermal amplification technique for Hepatitis B virus genotypes B and C genotyping and quantification

Cai, Zhejun; Lou, Guo-qiang; Cai, Ting; Yang, Jin; Wu, Nanping

doi:10.1016/j.jcv.2011.08.013

Cited by 20 publications

(12 citation statements)

References 30 publications

(33 reference statements)

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“…The linearity of quantification was evaluated using the 10-fold RNA copy panel (equal to 10 7 to 10 copies per reaction). The detection limit was determined by serial dilutions of lower numbers of copies (equal to 10,000, 5,000, 1,000, 500, 250, 100, 50, 25, 10, 5, and 1 copy per reaction) in 15 replicates, as described previously (29,30). The optimized one-tube real-time RT-LAMP assays established in our laboratory (18,23) were conducted as parallel tests, using the same amount and type of templates.…”

Section: Methodsmentioning

confidence: 99%

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

et al. 2014

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c Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-footand-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-selfmatching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R 2 values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R 2 values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses. Hand-foot-and-mouth disease (HFMD) is a childhood syndrome and is characterized by tiny blisters on the skin and oral mucosa together with fever and poor appetite. The two major causative agents of HFMD are human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). EV71-related HFMD commonly accounts for the fatal cases, due to severe complications, such as brainstem encephalitis and rapid fatal pulmonary edema, whereas CVA16-related HFMD usually presents with mild symptoms (1-3). Since no vaccine or antiviral drugs are currently available to treat HFMD, the early and rapid detection of EV71 and CVA16 are critical for the prevention and control of HFMD infection.Presently, laboratory detection of EV71 and CVA16 includes traditional virus isolation, neutralization, and nucleic acid amplification techniques. It is difficult to achieve rapid detection using traditional virus isolation and neutralization because of their low specificities and detection limits, as well as the long time that is spent obtaining results (4-6). Nucleic acid a...

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Section: Methodsmentioning

confidence: 99%

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

et al. 2014

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“…DBSs are the only available, viable option for HBV‐DNA testing in resource‐limited settings . A number of innovative platforms and technologies for HBV‐DNA quantification have been developed to be used in POCs and resources‐limited regions including microchip PCR‐based HBV‐DNA amplification devices, an isothermal amplification (LAMP) technology that avoids fluorescence reagents and a Nanoplasmonic Electrical field‐enhanced Resonating Device (NE2RD) . All these assays must be validated for broader dynamic ranges and in different HBV genotypes.…”

Section: Barriers To Hbv Treatmentmentioning

confidence: 99%

How to improve access to therapy in hepatitis B patients

Subic

Zoulim

2018

Liver International

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Despite the availability of a preventive vaccine and active antiviral treatments that stop disease progression and reduce the risk of hepatocellular carcinoma, hepatitis B is still a major public health problem. Only an estimated 10% of the 257 million people living with HBV have been diagnosed and as few as 1% are being adequately treated. Barriers to diagnosis and treatment include: (i) limited awareness and lack of knowledge about HBV infection and HBV‐related diseases; (ii) under‐diagnosis with insufficient screening and referral to care; (iii) limited treatment due to drug availability, costs, reimbursement policies and the need for long‐term or life‐long therapy. These barriers and the actions needed to improve access to treatment are strongly influenced by the prevalence of infection and affect middle‐high vs low‐middle income countries differently, where most HBV carriers are found. In high‐prevalence regions and low‐to middle‐income countries, the main challenges are availability and cost while in low‐prevalence regions and middle‐to high‐income countries low screening rates, public awareness, social stigma and discrimination play an important role. Overcoming these challenges on a global scale is a complex clinical and public health challenge and multilateral commitment from pharmaceutical companies, governments, funders and the research community is lacking. The new WHO 2016 Global Health Sector Strategy on viral hepatitis targets testing and treatment, suggesting that important but strong actions are needed from advocacy groups, scientific societies and funding agencies to foster awareness and access to cure.

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“…Results showed that the test elicited the maximal accuracy at 10 5 IU/mL. In addition, significant viremic samples were further analyzed for correlation with time to turbidity thresholds derived from turbidity-based LAMP assay [12]. Unfortunately, linear regression pattern was not found; therefore, we concluded that the time to turbidity threshold could not be used to quantify HBV viral load in turbidity-based LAMP assay.…”

Section: Evaluation With Clinical Samplesmentioning

confidence: 95%

“…The 2 × 10 5 IU/ mL cutoff value was clinically defined as significant viremia of which initiating an antiviral therapy was highly recommended [4]. Previous HBV LAMP development utilized the fluorescent dye to detect the amplified product [11,12] and the results were proven equally sensitive and specific to standard qPCR method. However, those fluorescent-based LAMP assays were also developed under qPCR equipment that was unavailable in point-of-care setting.…”

Section: Evaluation With Clinical Samplesmentioning

confidence: 99%

“…Although several HBV LAMP protocols have been described, limitations were still noted whenever applying to point of care. For example, the detection system in previous developments required a qPCR machine [11,12] that was unavailable in the resource-limited area. Also, it is our aim to deliberately avoid chromogenic or fluorogenic substrates addition in order to minimize the cost per reaction.…”

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confidence: 99%

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Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

et al. 2018

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Background Hepatitis B virus (HBV) has been the most prevalent blood-borne pathogen wherein utero transmission has still not been properly managed. Recent practice guidelines suggested that an antiviral drug should be administered to third-trimester pregnancies with significant viremia (>2 × 105 IU/mL). Objectives To develop a novel turbidity-based loop-mediated isothermal amplification (LAMP) coupled with heat treatment DNA extraction method that is a rapid, cost-effective, and feasible viral load assessment and could be applied to antenatal screening. Methods Primers and reagents were designed, turbidity-based platform and heat treatment method were added, and evaluated for optimal efficiency. Assay sensitivity was tested from serially diluted standard HBV DNA. Assay specificity was tested with six standard viral DNAs. Clinical samples were analyzed and the results were compared with those of quantitative polymerase chain reaction (qPCR) diagnostic records. Results The optimized condition was 60°C with no betaine, 1.4 mM deoxyribonucleotide triphosphates (dNTPs) and 6 mM of MgSO4 for 60 min. The assay accurately detected samples with standard HBV DNA at >2 × 105 IU/mL in both distilled water and spiked serum. Results can be interpreted within 31.48 ± 1.41 min in real-time turbidimeter. The amplification is exclusively specific to HBV, but not with the other six human-specific viruses. Moreover, the assay showed comparable performance within 95% confidence interval to the previously developed HBV LAMP toward clinical specimens. Conclusions This newly developed method was accurate, affordable, and flexible to further implementation to large-scale third-trimester pregnancy screening.

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Development of a novel genotype-specific loop-mediated isothermal amplification technique for Hepatitis B virus genotypes B and C genotyping and quantification

Cited by 20 publications

References 30 publications

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

How to improve access to therapy in hepatitis B patients

Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

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