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Isothermal amplifications - a comprehensive review on current methods

Glökler J, Lim TS, Ida J, Frohme M

Crit Rev Biochem Mol Biol, 2021 12;56(6):543-586.
PMID: 34263688 DOI: 10.1080/10409238.2021.1937927

The introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
An integrated lateral flow assay for effective DNA amplification and detection at the point of care

Choi JR, Hu J, Gong Y, Feng S, Wan Abas WA, Pingguan-Murphy B, et al.

Analyst, 2016 05 10;141(10):2930-9.
PMID: 27010033 DOI: 10.1039/c5an02532j

Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future.

Matched MeSH terms: Nucleic Acid Amplification Techniques*
Loop-mediated isothermal amplification (LAMP) test in the detection of uncomplicated malaria in pregnancy: a meta-analysis of diagnostic accuracy

Yon JLT, Htet NH, Naing C, Tung WS, Aung HH, Mak JW

Malar J, 2022 Dec 22;21(1):391.
PMID: 36550507 DOI: 10.1186/s12936-022-04419-9

BACKGROUND: Due to relatively low malaria parasitaemia in pregnancy, an appropriate field test that can adequately detect infections in pregnant women presenting with illness or for malaria screening during antenatal care is crucially important. The objective was to evaluate the diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for the detection of uncomplicated malaria in pregnancy.
METHODS: This was a meta-analysis of diagnostic accuracy. Relevant studies that assessed the diagnostic performance of LAMP for the detection of malaria in pregnancy were searched in health-related electronic databases including PubMed, Ovid, and Google Scholar. The methodological quality of the studies included was evaluated using the QUADAS-2 tool.
RESULTS: Of the 372 studies identified, eight studies involving 2999 pregnant women in five endemic countries that assessed the accuracy of LAMP were identified. With three types of PCR as reference tests, the pooled sensitivity of LAMP was 91% (95%CI 67-98%) and pooled specificity was 99% (95%CI 83-100%, 4 studies), and the negative likelihood ratio was 9% (2-40%). Caution is needed in the interpretation as there was substantial between-study heterogeneity (I2: 80%), and a low probability that a person without infection is tested negative. With microscopy as a reference, the pooled sensitivity of LAMP was 95% (95%CI 26-100%) and pooled specificity was 100% (95%CI 94-100%, 4 studies). There was a wide range of sensitivity and substantial between-study heterogeneity (I2: 83.5-98.4%). To investigate the source of heterogeneity, a meta-regression analysis was performed with covariates. Of these potential confounding factors, reference test (p: 0.03) and study design (p:0.03) had affected the diagnostic accuracy of LAMP in malaria in pregnancy. Overall, there was a low certainty of the evidence in accuracy estimates.
CONCLUSION: The findings suggest that LAMP is more sensitive than traditional tests used at facilities, but the utility of detecting and treating these low-density infections is not well understood. Due to the limited number of studies with bias in their methodological quality, variation in the study design, and different types of reference tests further research is likely to change the estimate. Well-conceived large prospective studies with blinding of the index test results are recommenced.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
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.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
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.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
Streptococcus gallolyticus infection: A neglected marker for colorectal cancer?

Bin Ismail CMKH, Bin Mohammad Aidid E, Binti Hamzah HA, Bin Shalihin MSE, Bin Md Nor A

Arab J Gastroenterol, 2023 Aug;24(3):163-167.
PMID: 37156704 DOI: 10.1016/j.ajg.2023.02.002

BACKGROUND AND STUDY AIMS: Colorectal cancer (CRC) is the second most common cancer in Malaysia and mostly detected at advanced stages due to lack of awareness of CRC symptoms and signs. CRC pathogenesis is multifactorial, and there is ambiguous evidence on association of Streptococcus gallolyticus infection with CRC that needs further attention. Thus, a case-control study was conducted to determine whether S. gallolyticus infection is a predictor for CRC occurrence among patients attending Sultan Ahmad Shah Medical Centre@IIUM (SASMEC@IIUM).
PATIENTS AND METHODS: A total of 33 stool samples from patients diagnosed with CRC and 80 from patients without CRC attending surgical clinic of SASMEC@IIUM were collected and analyzed with iFOBT test and PCR assay to detect S. gallolyticus.
RESULTS: In this study, the proportion of S. gallolyticus infection was higher among patients with CRC (48.5%) compared with the control group (20%). Univariate analysis shows that occult blood in stool, S. gallolyticus infection and family history were significantly associated with the development of CRC (P

Matched MeSH terms: Nucleic Acid Amplification Techniques
IQPA: isothermal nucleic acid amplification-based immunoassay using DNAzyme as the reporter system

Loh Q, Omar N, Glökler J, Lim TS

Anal Biochem, 2014 Oct 15;463:67-9.
PMID: 24972268 DOI: 10.1016/j.ab.2014.06.012

Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.

Matched MeSH terms: Nucleic Acid Amplification Techniques*
Fulltext Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification

Lau YL, Lai MY, Teoh BT, Abd-Jamil J, Johari J, Sam SS, et al.

PLoS One, 2015;10(9):e0138694.
PMID: 26384248 DOI: 10.1371/journal.pone.0138694

Dengue is usually diagnosed by isolation of the virus, serology or molecular diagnostic methods. Several commercial kits for the diagnosis of dengue are existing, but concerns have arisen regarding to the affordability and performance characteristics of these kits. Hence, the loop-mediated isothermal amplification (LAMP) is potentially ideal to be used especially in resource limited environments. Serum was collected from healthy donors and patients diagnosed with dengue infection. RNA extracted from the serum samples were tested by reverse-transcription-LAMP assay developed based on 3'-NCR gene sequences for DENV 1-4. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. Sensitivity and specificity of RT-LAMP results were calculated and compared to qRT-PCR and ELISA. RT-LAMP is highly sensitive with the detection limit of 10 RNA copies for all serotypes. Dengue virus RNA was detected in all positive samples using RT-LAMP and none of the negative samples within 30-45 minutes. With continuing efforts in the optimization of this assay, RT-LAMP may provide a simple and reliable test for detecting DENV in areas where dengue is prevalent.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Loop-mediated isothermal amplification (LAMP) test for diagnosis of uncomplicated malaria in endemic areas: a meta-analysis of diagnostic test accuracy

Selvarajah D, Naing C, Htet NH, Mak JW

Malar J, 2020 Jun 19;19(1):211.
PMID: 32560728 DOI: 10.1186/s12936-020-03283-9

BACKGROUND: The global malaria decline has stalled and only a few countries are pushing towards pre-elimination. The aim of the malaria elimination phase is interruption of local transmission of a specified malaria parasite in a defined geographical area. New and improved screening tools and strategies are required for detection and management of very low-density parasitaemia in the field. The objective of this study was to synthesize evidence on the diagnostic accuracy of loop-mediated isothermal amplification (LAMP) test for the detection of malaria parasites among people living in endemic areas.
METHODS: This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis for Diagnostic Test Accuracy (PRISMA-DTA) guideline. Relevant studies in the health-related electronic databases were searched. According to the criteria set for this study, eligible studies were identified. The quality of included studies was evaluated with the use of a quality assessment checklist. A summary performance estimates such as pooled sensitivity and specificity were stratified by type of LAMP. Bivariate model for data analyses was applied. Summary receiver operating characteristics plots were created to display the results of individual studies in a receiver operating characteristics space. Meta-regression analysis was performed to investigate the sources of heterogeneity among individual studies.
RESULTS: Twenty-seven studies across 17 endemic countries were identified. The vast majority of studies were with unclear risk of bias in the selection of index test. Overall, the pooled test performances were high for Pan LAMP (sensitivity: 0.95, 95% CI 0.91 to 0.97; specificity: 0.98, 95% CI 0.95 to 0.99), Plasmodium falciparum (Pf) LAMP (sensitivity: 0.96, 95% CI 0.94 to 0.98; specificity: 0.99, 95% CI 0.96 to 1.00) or for Plasmodium vivax (Pv) LAMP from 6 studies (sensitivity: 0.98, 95% CI 0.92 to 0.99; specificity: 0.99, 95% CI 0.72 to 1.00). The area under the curve for Pan LAMP (0.99, 95% CI 0.98-1.00), Pf LAMP (0.99, 95% CI 0.97-0.99) and Pv LAMP was (1.00, 95% CI 0.98-1.00) indicated that the diagnostic performance of these tests were within the excellent accuracy range. Meta-regression analysis showed that sample size had the greatest impact on test performance, among other factors.
CONCLUSIONS: The current findings suggest that LAMP-based assays are appropriate for detecting low-level malaria parasite infections in the field and would become valuable tools for malaria control and elimination programmes. Future well-designed larger sample studies on LAMP assessment in passive and active malaria surveillances that use PCR as the reference standard and provide sufficient data to construct 2 × 2 diagnostic table are needed.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Recombinase Polymerase Amplification Combined with a Lateral Flow Strip for the Detection of Plasmodium knowlesi

Lai MY, Ooi CH, Lau YL

Am J Trop Med Hyg, 2018 03;98(3):700-703.
PMID: 29260656 DOI: 10.4269/ajtmh.17-0738

The aim of this study was to develop a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method for specific diagnosis of Plasmodium knowlesi. With incubation at 37°C, the 18S rRNA gene of P. knowlesi was successfully amplified within 12 minutes. By adding a specifically designed probe to the reaction solution, the amplified RPA product can be visualized on a LF strip. The RPA assay exhibited high sensitivity with limits of detection down to 10 parasites/μL of P. knowlesi. Nonetheless, it was demonstrated that all P. knowlesi (N = 41) and other Plasmodium sp. (N = 25) were positive while negative samples (N = 8) were negative. Therefore, a combination of RPA and LF strip detection is a highly promising approach with the potential to be suitable for use in resource-limited settings.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Long Fragment Polymerase Chain Reaction

Chua EW, Maggo S, Kennedy MA

Methods Mol Biol, 2017;1620:65-74.
PMID: 28540699 DOI: 10.1007/978-1-4939-7060-5_3

Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Rapid electrochemical detection of coronavirus SARS-CoV-2

Chaibun T, Puenpa J, Ngamdee T, Boonapatcharoen N, Athamanolap P, O'Mullane AP, et al.

Nat Commun, 2021 02 05;12(1):802.
PMID: 33547323 DOI: 10.1038/s41467-021-21121-7

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
Fulltext Malaria diagnosis using a combined system of a simple and fast extraction method with a lyophilised Dual-LAMP assay in a non-endemic setting

Martín Ramírez A, Barón Argos L, Lanza Suárez M, Carmona Rubio C, Pérez-Ayala A, Hisam SR, et al.

Pathog Glob Health, 2024 Feb;118(1):80-90.
PMID: 37415348 DOI: 10.1080/20477724.2023.2232595

Malaria is a parasitic disease distributed in tropical areas but with a high number of imported cases in non-endemic countries. The most specific and sensitive malaria diagnostic methods are PCR and LAMP. However, both require specific equipment, extraction procedures and a cold chain. This study aims to solve some limitations of LAMP method with the optimization and validation of six LAMP assays, genus and species-specific, using an easy and fast extraction method, the incorporation of a reaction control assay, two ways (Dual) of result reading and reagent lyophilization. The Dual-LAMP assays were validated against the Nested-Multiplex Malaria PCR. A conventional column and saline extraction methods, and the use of lyophilized reaction tubes were also assessed. A new reaction control Dual-LAMP-RC assay was designed. Dual-LAMP-Pspp assay showed no cross-reactivity with other parasites, repeatability and reproducibility of 100%, a significant correlation between parasite concentration and time to amplification and a LoD of 1.22 parasites/µl and 5.82 parasites/µl using column and saline extraction methods, respectively. Sensitivity and specificity of the six Dual-LAMP assays reach values of 100% or close to this, being lower for the Dual-LAMP-Pm. The Dual-LAMP-RC assay worked as expected. Lyophilized Dual-LAMP results were concordant with the reference method. Dual-LAMP malaria assays with the addition of a new reaction control LAMP assay and the use of a fast and easy saline extraction method, provided low limit of detection, no cross-reactivity, and good sensitivity and specificity. Furthermore, the reagent lyophilization and the dual result reading allow their use in most settings.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
Fulltext Real-time PCR evaluation of seven DNA extraction methods for the purpose of GMO analysis

Jasbeer, K., Son, R., Mohamad Ghazali, F., Cheah, Y.K.

International Food Research Journal, 2009;16(3):329-341.
MyJurnal

Successful DNA amplification is vital for the detection of specific DNA targets in feeds, and this in return depends on the ability of DNA extraction methods to produce good quality DNA. In this study, seven methods were compared for DNA extraction from feeds using quantitative polymerase chain reaction (PCR) of single copy maize (Zea mays) endogenous hmg (high mobility group) gene. Relative levels of hmg were used to evaluate the DNA quality. Spectrophotometer determination of DNA was also carried out to assess DNA yield and DNA purity, while electrophoretic analysis of genomic DNA extracts was carried out to investigate DNA integrity. The findings illustrate that the DNA extraction methods have a significant effect on DNA quality. Statistically, the Epicentre method extracted the highest DNA yield while the Wizard method had the lowest DNA yield with high DNA purity and integrity. However, the Wizard method recovered the most amplifiable DNA per reaction, indicating that template quality and integrity had greater influence over hmg amplification than DNA yield.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Development of the compact UV illuminator with single UV lamp

Lee DJ, Kim SY, Kim JD, Kim YS, Song HJ, Park CY

Sains Malaysiana, 2015;44:1693-1699.

This paper presents a low-cost method of constructing the compact UV illuminator, which is considered as an important
component of a gel documentation system. The procedure involves using a smallest-possible UV lamp and a motor which
moves the UV lamp in the UV illuminator instead of conventional 4 UV lamps. A comparative analysis of images produced
by using the commercial gel documentation system and our prototype was carried out. These comparisons were done
in real DNA gel as well as a reference plate made of quantum dot. The plate was composed of the chambers filled with
various densities of the quantum dot instead of the Agarose gel containing the ETBR in order to increase the accuracy of
comparison and the convenience of experiments. Despite the use of only 1 UV lamp, the proposed system demonstrated
a similar imaging performance compared with the conventional gel documentation system equipped with 4 UV lamps,
resulting in the great reduction of the system cost.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Clinical relevance of molecular testing methods in the diagnosis and guidance of therapy in patients with staphylococcal empyema: a systematic review and meta-analysis

Ojha SC, Chen K, Yuan Y, Ahmed S, Malik AA, Nisha M, et al.

Front Cell Infect Microbiol, 2022;12:758833.
PMID: 35967859 DOI: 10.3389/fcimb.2022.758833

BACKGROUND: Efficient detection tools for determining staphylococcal pleural infection are critical for its eradication. The objective of this meta-analysis was to assess the diagnostic utility of nucleic acid amplification tests (NAAT) in suspected empyema cases to identify staphylococcal strains and avoid unnecessary empiric methicillin-resistant Staphylococcus aureus (MRSA) therapy.
METHODS: From inception to July 24, 2021, relevant records were retrieved from PubMed, Embase, Scopus, Web of Science, and the Cochrane Library. The quality of studies was determined using the QUADAS-2 tool. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and hierarchical summary receiver operating characteristic (HSROC) curve for NAAT's diagnostic performance were evaluated using an HSROC model.
RESULTS: Eight studies comprising 424 samples evaluated NAAT accuracy for Staphylococcus aureus (SA) identification, while four studies comprising 317 samples evaluated methicillin-resistant Staphylococcus aureus (MRSA) identification. The pooled NAAT summary estimates for detection of both SA (sensitivity: 0.35 (95% CI 0.19-0.55), specificity: 0.95 (95% CI 0.92-0.97), PLR: 7.92 (95% CI 4.98-12.59), NLR: 0.44 (95% CI 0.14-1.46), and DOR: 24.0 (95% CI 6.59-87.61) ) and MRSA (sensitivity: 0.45 (95% CI 0.15-0.78), specificity: 0.93 (95% CI 0.89-0.95), PLR: 10.06 (95% CI 1.49-67.69), NLR: 0.69 (95% CI 0.41-1.15), and DOR: 27.18 (95% CI 2.97-248.6) ) were comparable. The I2 statistical scores for MRSA and SA identification sensitivity were 13.7% and 74.9%, respectively, indicating mild to substantial heterogeneity. PCR was frequently used among NAA tests, and its diagnostic accuracy coincided well with the overall summary estimates. A meta-regression and subgroup analysis of country, setting, study design, patient selection, and sample condition could not explain the heterogeneity (meta-regression P = 0.66, P = 0.46, P = 0.98, P = 0.68, and P = 0.79, respectively) in diagnostic effectiveness.
CONCLUSIONS: Our study suggested that the diagnostic accuracy of NAA tests is currently inadequate to substitute culture as a principal screening test. NAAT could be used in conjunction with microbiological culture due to the advantage of faster results and in situations where culture tests are not doable.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext A Sensitive Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Direct Visual Detection of SARS-CoV-2

Lau YL, Ismail IB, Izati Binti Mustapa N, Lai MY, Tuan Soh TS, Hassan AH, et al.

Am J Trop Med Hyg, 2020 Dec;103(6):2350-2352.
PMID: 33098286 DOI: 10.4269/ajtmh.20-1079

A simple and rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of SARS-CoV-2. The RT-LAMP assay was highly specific for SARS-CoV-2 and was able to detect one copy of transcribed SARS-CoV-2 RNA within 24 minutes. Assay validation performed using 50 positive and 32 negative clinical samples showed 100% sensitivity and specificity. The RT-LAMP would be valuable for clinical diagnosis and epidemiological surveillance of SARS-CoV-2 infection in resource-limited areas as it does not require the use of sophisticated and costly equipment.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods; Nucleic Acid Amplification Techniques/standards*
Fulltext Colorimetric Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2

Lai MY, Tang SN, Lau YL

Am J Trop Med Hyg, 2021 Jun 15;105(2):375-377.
PMID: 34129521 DOI: 10.4269/ajtmh.21-0150

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly all over the world. In the absence of effective treatments or a vaccine, there is an urgent need to develop a more rapid and simple detection technology of COVID-19. We describe a WarmStart colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2. The detection limit for this assay was 1 copy/µL SARS-CoV-2. To test the clinical sensitivity and specificity of the assay, 37 positive and 20 negative samples were used. The WarmStart colorimetric RT-LAMP had 100% sensitivity and specificity. End products were detected by direct observation, thereby eliminating the need for post-amplification processing steps. WarmStart colorimetric RT-LAMP provides an opportunity to facilitate virus detection in resource-limited settings without a sophisticated diagnostic infrastructure.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*; Nucleic Acid Amplification Techniques/standards
Evaluation of serological transfusion-transmitted viral diseases and mutliplex nucleic acid testing in malaysian blood donors

Yaseen SG, Ahmed SA, Johan MF, Kiron R, Daher AM

Transfus Apher Sci, 2013 Dec;49(3):647-51.
PMID: 23890575 DOI: 10.1016/j.transci.2013.07.003

Transmission of infectious diseases is a recognized complication of blood transfusion and blood products. Nucleic acid testing (NAT) may contribute to improved efficiency of blood screening and thereby increase the safety margin for transfused blood.

Matched MeSH terms: Nucleic Acid Amplification Techniques/instrumentation; Nucleic Acid Amplification Techniques/methods*
A shelf-stable fluorogenic isothermal amplification assay for the detection of Burkholderia pseudomallei

Michelle Wong Tzeling J, Yean Yean C

Analyst, 2016 Feb 21;141(4):1246-9.
PMID: 26783560 DOI: 10.1039/c5an01741f

A shelf-stable loop-mediated isothermal amplification (LAMP) reagent for Burkholderia pseudomallei detection is described. The coupling of LAMP reagents with the indirect colorimetric indicator and consequently its lyophilization enable the simple evaluation of results without the need for any advance laboratory instruments. The reagents were found to have a stable shelf life of at least 30 days with well-maintained sensitivity and specificity.

Matched MeSH terms: Nucleic Acid Amplification Techniques

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