Sequence‐specific detection method for reverse transcription, loop‐mediated isothermal amplification of HIV‐1

Curtis, Kelly A.; Rudolph, Donna L.; Owen, S. Michele

doi:10.1002/jmv.21490

Cited by 67 publications

(75 citation statements)

References 30 publications

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“…The RT-LAMP assay from this study and other studies [17] can therefore be presented as devoid of cross-reactivity with other viruses and parasites such as malaria parasite. It is however important to note that although this study and others mentioned for HCV have not reported cross-reactivity, other studies done with RT-LAMP [12,24] have indicated a potential for non-specific binding leading to false positive results. In their work Odari and the group [12] suggests that the complex reaction process coupled by the number of primers used for any LAMP reaction are a potential source of unspecific binding leading to the generation of Magnesium pyrophosphate (Mg 3 (PO 4 ) 2 ) deposits generated by detectable by the turbidimetric reading.…”

Section: /9contrasting

confidence: 50%

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

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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.

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Section: /9contrasting

confidence: 50%

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

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“…As a result, this technology has been used widely for molecular detection of several microorganisms, including clinical and plant-associated bacterial [32,[61][62][63][64][65][66][67][68][69][70][71][72][73], fungal [74][75][76], viral [47,[77][78][79][80][81][82][83][84], and parasitic [57,59,[85][86][87][88] pathogens, and is therefore suitable as a rapid field test.…”

Section: Discussionmentioning

confidence: 99%

Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

Yasuhara-Bell¹,

Ayin²,

Hatada³

et al. 2015

J Plant Pathol Microbiol

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Klebsiella spp. are opportunistic pathogens with clinical, veterinary and plant-associated isolates. A previous study showed that bacterial ooze from wetwood of severely declined ironwood trees in Guam contained Ralstonia solanacearum, Klebsiella variicola and K. oxytoca. In this study, Loop-Mediated Isothermal Amplification (LAMP) detected K. variicola and K. oxytoca specifically, using unique primer sets designed individually for each organism. Each LAMP detected its target specifically, while showing negative results for non-target bacteria and negative controls. LAMP detected Klebsiella in inoculated-ironwood stem tissues and bacterial ooze. Due to the presence of plant inhibitors, different sampling protocols were tested. Soaking plant tissue samples to allow diffusion of bacteria into solution, followed by boiling, provided optimum detection of Klebsiella directly from plant samples. False negatives obtained when using crushed plant samples were eliminated by including an enrichment step, involving plating and 12-h incubation. DGGE (denaturing gradient gel electrophoresis) and a colony-blot immunoassay using a Klebsiella-specific antibody also detected Klebsiella in inoculated ironwood. DGGE bands and antibody crossreactions from closely related enterobacters showed the potential for false positive results. The nature of LAMP makes it ideal for point-of-care testing, and when combined with the specificity of the LAMP primers developed in this study, demonstrates its potential as a routine field test for Klebsiella in ironwood in Guam, as well as clinical and veterinary diagnosis of Klebsiella infection. Additionally, the regions targeted for detection in this study have application across all forms of molecular-based diagnostics.

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“…Visual or photographic assessment of fluorescence originated by FRET probes have been used for specific detection of LAMP products (Curtis et al 2009). For unspecific detection of LAMP products, diverse authors have used intercalating dyes like SYBR green or monitored the changes in turbidity of the reaction originated by the precipitation of magnesium pyrophosphate (Mori et al 2001) due to large amount of pyrophosphate originated in the polymerization reaction.…”

Section: Lamp As An Alternative To Pcrmentioning

confidence: 99%

“…A lateral flow device has also been used for the detection of amplified products (Rigano et al 2010). However, some occasional reports of unspecific amplifications (see Curtis et al (2009)) may restrain the enthusiasm in the development of unspecific methods for monitoring the amplification.…”

Section: Lamp As An Alternative To Pcrmentioning

confidence: 99%

New Developments in Pathogen Detection and Identification

Nolasco¹

2012

Acta Hortic.

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A b s tractBy the turn of the century most of the methods for amplifying the pathogen detection signal had already been developed. However only a few were successfully incorporated into routine diagnosis schemes. At present, real time PCR became the predominant choice with signal transduction based on fluorescence generated by intercalating dyes or fluorescent resonant energy transfer. Real-time PCR is no more exclusively restricted to niches for which no alternative was feasible; its use is widening and is substituting or complementing other techniques like ELISA. On the low technology side and judging from the success in medical fields, Loop Mediated Isothermal Amplification (LAMP) appears as an interesting alternative. Convergence into a single platform led to the concept of crop-oriented diagnosis, e.g, a unique device or system that could be used in the detection of the relevant pathogens of a crop. However PCR by itself is not enough robust to support the degree of multiplexing needed and fluorescence detection systems are limited to a reduced number of dyes that can be used simultaneously. Initial attempts to develop alternative systems relied in the use of microarrays but suffer from limited sensitivity. Low Density Arrays, in which individual PCR reactions are spatially separated and done in parallel appear an interesting option, already available for grapevine. All these assays only provide an answer regarding the presence of certain pathogens for which there is an a priori suspicion. Recently introduced high massively parallel sequencing coupled to metagenomic analysis appears to be a major breakthrough in diagnosis, enabling the non-targeted diagnosis of bacteria, virus, fungi and novel agents in a single assay. These have been implemented among others for citrus and grapevine.

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Sequence‐specific detection method for reverse transcription, loop‐mediated isothermal amplification of HIV‐1

Cited by 67 publications

References 30 publications

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

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

Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

New Developments in Pathogen Detection and Identification

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