MyMedR

Fulltext Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.

BMC Infect Dis, 2021 Nov 17;21(1):1162.

PMID: 34789179 DOI: 10.1186/s12879-021-06876-0

BACKGROUND: Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step.

METHODS: In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets.

RESULTS: Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9-96.1%) and 67.4% sensitive (95% CI: 51.5-80.9%) for E gene and RdRp gene, respectively.

CONCLUSION: Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

Fulltext Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Lau YL, Ismail IB, Mustapa NIB, Lai MY, Tuan Soh TS, Haji Hassan A, et al.

PLoS One, 2021;16(1):e0245164.

PMID: 33406112 DOI: 10.1371/journal.pone.0245164

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

Fulltext RNA purification-free detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Lai MY, Suppiah J, Thayan R, Ismail I, Mustapa NI, Soh TST, et al.

Trop Med Health, 2022 Jan 04;50(1):2.

PMID: 34980275 DOI: 10.1186/s41182-021-00396-y

BACKGROUND: Current diagnosis of SARS-CoV-2 infection relies on RNA purification prior to amplification. Typical extraction methods limit the processing speed and turnaround time for SARS-CoV-2 diagnostic testing.

METHODS: Here, we applied reverse transcription loop-mediated isothermal amplification directly onto human clinical swabs samples to amplify the RNA from SARS-CoV-2 swab samples after processing with chelating resin.

RESULTS: By testing our method on 64 samples, we managed to develop an RT-LAMP assay with 95.9% sensitivity (95% CI 86 to 99.5%) and 100% specificity (95% CI 78.2-100%).

CONCLUSION: The entire process including sample processing can be completed in approximately 50 min. This method has promising potential to be applied as a fast, simple and inexpensive diagnostic tool for the detection of SARS-CoV-2.

Fulltext Colorimetric detection of SARS-CoV-2 by uracil-DNA glycosylase (UDG) reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.

Int J Infect Dis, 2022 Jul;120:132-134.

PMID: 35472524 DOI: 10.1016/j.ijid.2022.04.036

OBJECTIVES: Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix.

METHODS: RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method.

RESULTS: Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78-98.2%) and 100% specificity (95% confidence interval: 92-100%) using UDG RT-LAMP.

CONCLUSION: This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities.

Fulltext Two-Stage Detection of Plasmodium spp. by Combination of Recombinase Polymerase Amplification and Loop-Mediated Isothermal Amplification Assay

Lai MY, Lau YL

Am J Trop Med Hyg, 2022 Oct 12;107(4):815-819.

PMID: 35970289 DOI: 10.4269/ajtmh.22-0136

We developed a combination of recombinase polymerase and loop-mediated isothermal amplification methods (RAMP) for rapid screening of five human Plasmodium spp. simultaneously. RAMP is a two-stage isothermal amplification method, which consists of a first-stage recombinase polymerase amplification and a second-stage loop-mediated isothermal amplification. Under these two isothermal conditions, five Plasmodium spp. were amplified in less than 40 minutes. We demonstrated RAMP assay with 10-fold better limit of detection than a single (loop-mediated isothermal amplification) LAMP. As compared with microscopy, RAMP assay showed 100% sensitivity (95% CI: 95.65-100.00%) and 100% specificity (95% CI: 69.15-100.00%). The end products were inspected by the color changes of neutral red. Positive reactions were indicated by pink while the negative reactions remained yellow. The combination assay established in this study can be used as a routine diagnostic method for malaria.

Lateral Flow Recombinase Polymerase Amplification Assays for the Detection of Human Plasmodium Species

Lai MY, Abdul Hamid M, Jelip J, Mudin RN, Lau YL

Am J Trop Med Hyg, 2023 Mar 13.

PMID: 36913921 DOI: 10.4269/ajtmh.22-0657

This study highlights the development of two lateral flow recombinase polymerase amplification assays for the diagnosis of human malaria. The lateral flow cassettes contained test lines that captured biotin-, 6-carboxyfluorescein, digoxigenin-, cyanine 5-, and dinitrophenyl-labeled amplicons. The overall process can be completed in 30 minutes. Recombinase polymerase amplification coupled with lateral flow had a detection limit of 1 copy/µL for Plasmodium knowlesi, Plasmodium vivax, and Plasmodium falciparum. No cross-reactivity was observed among nonhuman malaria parasites such as Plasmodium coatneyi, Plasmodium cynomolgi, Plasmodium brasilanium, Plasmodium inui, Plasmodium fragile, Toxoplasma gondii, Sarcocystis spp., Brugia spp., and 20 healthy donors. It is rapid, highly sensitive, robust, and easy to use. The result can be read without the need for special equipment and thus has the potential to serve as an effective alternative to polymerase chain reaction methods for the diagnosis of malaria.

Fulltext Correction: Validation of SYBR green I based closed-tube loop-mediated isothermal amplification (LAMP) assay for diagnosis of knowlesi malaria

Lai MY, Ooi CH, Lau YL

Malar J, 2023 Oct 18;22(1):314.

PMID: 37853443 DOI: 10.1186/s12936-023-04731-y

Correction: Plasmodium knowlesi: the game changer for malaria eradication

Lee WC, Cheong FW, Amir A, Lai MY, Tan JH, Phang WK, et al.

Malar J, 2023 Oct 19;22(1):316.

PMID: 37858164 DOI: 10.1186/s12936-023-04732-x

Fulltext Corrigendum to "Colorimetric detection of SARS-CoV-2 by uracil-DNA glycosylase (UDG) reverse transcription loop-mediated isothermal amplification (RT-LAMP)" [International Journal of Infectious Diseases 120 (2023) Pages 132-134]

Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.

Int J Infect Dis, 2023 Nov;136:91.

PMID: 37774600 DOI: 10.1016/j.ijid.2023.09.011

Correction: RNA purification-free detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Lai MY, Suppiah J, Thayan R, Ismail I, Mustapa NI, Soh TST, et al.

Trop Med Health, 2023 Nov 22;51(1):66.

PMID: 37993919 DOI: 10.1186/s41182-023-00555-3

Corrigendum to: "Detection of Plasmodium knowlesi using recombinase polymerase amplification (RPA) combined with SYBR Green I" [volume 208, article number ACTROP_105511]

Lai MY, Lau YL

Acta Trop, 2023 Dec;248:107029.

PMID: 37777354 DOI: 10.1016/j.actatropica.2023.107029

A visualized hybrid PCR-LAMP assay for the detection of human Plasmodium species

Lai MY, Ponnampalavanar SSS, Omar SFS, Lau YL

Acta Trop, 2024 Mar;251:107120.

PMID: 38199452 DOI: 10.1016/j.actatropica.2024.107120

Combining the advantages of PCR and LAMP, we described a new technique, namely PCR-LAMP, for malaria diagnosis. The whole process of DNA amplification can be completed in 35 min. This hybrid amplification technique markedly improved the sensitivity of detection compared to the classic single PCR or LAMP assay alone. PCR-LAMP assay had a detection limit of 1 copy/µL for P. knowlesi and P. ovale, 0.1 copy/µL for P. vivax, P. falciparum and P. malariae, respectively. To facilitate the endpoint detection, xylenol orange was added. Positive samples were indicated in orange while negative reactions were violet. The inclusion of xylenol orange into the LAMP reaction mix significantly reduces the post-amplification workload. Without relying on the use of specific instruments, the color changes of the amplicons could be visualized directly through the naked eye. In conclusion, PCR-LAMP poses the potential to be developed as a new malaria molecular diagnosis tool.

Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi

Lai MY, Abdul Hamid MH, Jelip J, Mudin RN, Lau YL

Am J Trop Med Hyg, 2024 Apr 03;110(4):648-652.

PMID: 38412548 DOI: 10.4269/ajtmh.23-0572

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.

Displaying publications 21 - 33 of 33 in total