Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus

Kargar, Mohammad; Askari, Ahzam; Doosti, Abbas; Ghorbani-Dalini, Sadegh

doi:10.1007/s13337-012-0067-2

Cited by 24 publications

(27 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[7b] In this work, we used RT-LAMP primers (see Table S1 in Supporting Information (SI)) modified from previous work targeting the conserved 5′-untranslated region (5′UTR) of HCV. [8] RE-based digestion is a reliable method to recognize specific nucleic acid sequences of multiple letters in length and cleave at specific sites. [9] We hypothesized that RE digestion could be used to compete with RT-LAMP amplification in situ in both bulk and digital formats.…”

mentioning

confidence: 99%

Measuring Fate and Rate of Single‐Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA

Sun¹,

Rodríguez-Manzano²,

Selck³

et al. 2014

Angew Chem Int Ed

View full text Add to dashboard Cite

We experimentally monitored, at the single-molecule level, the competition among reverse transcription, exponential amplification (RT-LAMP), and linear degradation (restriction enzymes) starting with Hepatitis C viral RNA molecules. We found significant heterogeneity in the rate of single-molecule amplification; introduction of the restriction enzymes affected both the rate and the “fate” (the binary outcome) of single-molecule amplification. While end-point digital measurements were primarily sensitive to changes in fate, the bulk real-time kinetic measurements were dominated by the rate of amplification of the earliest molecules, and not sensitive to fate of the rest of the molecules. We showed how this competition of reactions can be used for rapid HCV genotyping with either digital or bulk readout. This work advances our understanding of single-molecule dynamics in reaction networks and may help bring genotyping capabilities out of clinical labs and into limited-resource settings.

show abstract

mentioning

confidence: 99%

Measuring Fate and Rate of Single‐Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA

Sun¹,

Rodríguez-Manzano²,

Selck³

et al. 2014

Angew Chem Int Ed

View full text Add to dashboard Cite

show abstract

“…Clinical evaluations of the C. difficile and group A Streptococcus tests have demonstrated sensitivity and specificity similar to those of traditional real-time PCR, though a slight decrease in sensitivity for C. difficile has been noted (39)(40)(41)(42)(43)(44). Laboratory-developed and commercially available research-use-only (RUO) tests using LAMP have targeted diverse groups of microorganisms, including Plasmodium spp., Giardia lamblia, Leishmania, Mycobacterium spp., and hepatitis viruses (45)(46)(47)(48)(49)(50). Specifically, LAMP-based testing for Plasmodium spp.…”

Section: Singleplex Nucleic Acid Testsmentioning

confidence: 99%

Emerging Technologies for the Clinical Microbiology Laboratory

2014

View full text Add to dashboard Cite

SUMMARY In this review we examine the literature related to emerging technologies that will help to reshape the clinical microbiology laboratory. These topics include nucleic acid amplification tests such as isothermal and point-of-care molecular diagnostics, multiplexed panels for syndromic diagnosis, digital PCR, next-generation sequencing, and automation of molecular tests. We also review matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry methods and their role in identification of microorganisms. Lastly, we review the shift to liquid-based microbiology and the integration of partial and full laboratory automation that are beginning to impact the clinical microbiology laboratory.

show abstract

“…Whereas the LAMP primers used had previously been described [16], in-house primers with a few modifications to include wobble bases were generated and used during the RT-PCR amplification. These primers for RT PCR were designated as outer primers; HCVN 01-GGC GAC ACT CCA CCA TRR A (forward), HCVN 02 -GTG CAC GGT CTA CGA GAC C (reverse), HCVN 08 -TAC TCA CCG GTT CCG CAG A (Reverse), Inner primers HCVN 03 -CAC TCC CCT GTG AGG AAC T -3' (forward), HCVN 04 -CCC GGG GCA CTC GCA AGC A (Reverse) with HCVN 02 and HCVN 08 being used during reverse transcription to generate HCV cDNA.…”

Section: Reaction Primersmentioning

confidence: 99%

“…Although a study [17] has evaluated LAMP on genotypes 1-6, a number of studies published [16,18] have dwelt mainly on genotypes circulating in Asian countries and the Arab world. This study presents the success of RT-LAMP assay under limited logistical conditions and further shows the differences in amplification of LAMP on a selected genotypes 1a, 1b, mixed 1a/1b, 2b, 3a and 4.…”

Section: /9mentioning

confidence: 99%

See 1 more Smart Citation

Evaluation of A Reverse Transcriptase (Rt) Loop Mediated Isothermal Amplification Assay for Detection of Hepatitis C Genotypes 1-4 Viruses Under Limited Logistical Conditions

Odari¹

2017

JHVRV

View full text Add to dashboard Cite

The loop-amplification mediated isothermal amplification (LAMP) presents characteristics that overcome the limitations associated with the current nucleic acid based technologies (NATs), hindering their use in the low resource settings, hence overreliance on serological assays which miss hepatitis C virus (HCV) during the long seroconversion period. The LAMP assay would be ideal for early detection of HCV in routine diagnostics and blood transfusion setups in such settings, drastically reducing transmission related transfusion. This study validated and tested a reverse transcriptase-LAMP for HCV detection under limited logistical conditions in a low resource setting.Under stringent laboratory conditions, analytical sensitivity and reproducibility were performed using a panel of HCV positive plasma of genotypes 1a, 1b, mixed 1a/1b, 2b, 3a and 4. Cell culture supernatants of HIV-1 B and plasma samples for Hepatitis B virus were used for specificity testing. Upto 227 plasma including 70 (40 RNA positive and 30 negative) from German patients and 157 (43 RNA positive and 114 negative) from Kenyan patients were tested. Kenyan samples were obtained from 121 sero-positive plasma screened from 1121 participants of various cohorts in Kenya.Although LAMP detected upto 102 IU/mL for genotypes 1a,1b and 2b, a lower detection threshold was established at 103 IU/mL. Overall sensitivity was 94% (PPV 98%) and specificity was 98% (NPV 96%) for RT-LAMP. Sub optimal detection was noted for genotypes 2b, 3a and 4. Sequence analysis revealed mismatches affecting stringency of primer binding at the F1, B1 and the LB primer targets.RT-LAMP shows potential for early HCV diagnosis and screening in low resource settings. Its robustness is however genotype dependent and can be enhanced by designing primers targeting circulating and suspected genotypes. More studies should be done on the possibility of designing multiplex RT-LAMP primers to capture a wide variety of genotypes. The assay remains simple, rapid, and cost effective for nucleic acid detection and is ideal for use in the limited resource settings.

show abstract

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus

Cited by 24 publications

References 24 publications

Measuring Fate and Rate of Single‐Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA

Measuring Fate and Rate of Single‐Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA

Emerging Technologies for the Clinical Microbiology Laboratory

Evaluation of A Reverse Transcriptase (Rt) Loop Mediated Isothermal Amplification Assay for Detection of Hepatitis C Genotypes 1-4 Viruses Under Limited Logistical Conditions

Contact Info

Product

Resources

About