A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood

Curtis, Kelly A.; Morrison, Daphne; Rudolph, Donna L.; Shankar, Anupama; Bloomfield, Laura S. P.; Switzer, William M.; Owen, S. Michele

doi:10.1016/j.jviromet.2018.02.012

Cited by 61 publications

(54 citation statements)

References 39 publications

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“…It represents a crucial strategy for parasite load determination in clinical samples, and allows the number of parasites to be normalized by the DNA quantity present in the sample [15]. For human patients, the RNAse P is a commonly used target as an internal control of reaction [56][57][58][59][60][61][62]. In Leishmania however, there are no studies, except the present one, describing the use of an internal control to quantify parasite load from human patient's samples.…”

Section: Discussionmentioning

confidence: 99%

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Filgueira¹,

Moreira²,

Cantanhêde³

et al. 2020

PLoS Negl Trop Dis

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Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.

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Section: Discussionmentioning

confidence: 99%

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Filgueira¹,

Moreira²,

Cantanhêde³

et al. 2020

PLoS Negl Trop Dis

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“…Results obtained after a 40-min incubation are provided in Figure 2(c). When these results were compared to RT-qPCR Ct values obtained from the same patient samples, it was determined that RT-LAMP was most sensitive in terms of detecting positive patients with viral loads that corresponds to low and medium Note: GeneN-A primers were described by Zhang et al 4 RNase P POP7 primers were described by Curtis et al 9 RT-qPCR Ct values. For Ct values under 28.8, the TP rate reported by RT-LAMP was 93%.…”

Section: Applying Rt-lamp To a Cohort Of 83 Suspected Sars-cov2 Patientsmentioning

confidence: 99%

Direct on-the-spot detection of SARS-CoV-2 in patients

Ben-Assa

Naddaf

Gefen

et al. 2020

Exp Biol Med (Maywood)

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Many countries are currently in a state of lockdown due to the SARS-CoV-2 pandemic. One key requirement to safely transition out of lockdown is the continuous testing of the population to identify infected subjects. Currently, detection is performed at points of care using quantitative reverse-transcription PCR, thus requiring dedicated professionals and equipment. Here, we developed a protocol based on reverse transcribed loop-mediated isothermal amplification for the detection of SARS-CoV-2. This protocol is applied directly to SARS-CoV-2 nose and throat swabs, with no RNA purification step required. We tested this protocol on over 180 suspected patients, and compared the results to those obtained using the standard method. We further succeeded in applying the protocol to self-collected saliva samples from confirmed cases. Since the proposed protocol can detect SARS-CoV-2 from saliva and provides on-the-spot results, it allows simple and continuous surveillance of the community. Impact statement Humanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.

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“…From this point forward, we also include a control RT-LAMP primer set, "RNaseP," which amplifies the humanderived mRNA produced from the POP7 gene, and therefore serves as a positive control that the reaction worked. This primer set was developed in 2018 (Curtis et al, 2018).…”

Section: Targeting Thementioning

confidence: 99%

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

Yang

Meyerson

Clark

et al. 2020

Preprint

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Up to 70% of SARS-CoV-2 infections in working- and school-age people are asymptomatic (Poletti et al., 2020), creating anxiety over reopening workplaces and schools around the world. In the absence of effective treatments or a vaccine, peace of mind will come only with community-based SARS-CoV-2 screening, where many people are tested on a regular basis. However, recent models show that short sample-to-answer turnaround time will be a critical property of effective screening strategies (Larremore et al., 2020). Here, we describe an RT-LAMP test for SARS-CoV-2 in raw saliva that takes about 45 minutes from sample to answer and requires only simple equipment (pipettes and a heating source). The assay has a limit of detection of 100 virions per microliter, and targets two separate regions of the SARS-CoV-2 genome. By combining rapid sample-to-answer turnaround time with the use of saliva, our RT-LAMP assay provides a low-complexity, portable, and robust system for real-time community screening.

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A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood

Cited by 61 publications

References 39 publications

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Direct on-the-spot detection of SARS-CoV-2 in patients

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

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