Reverse Transcription Loop-mediated Isothermal Amplification Method Research Articles

A colorimetric reverse-transcription loop-mediated isothermal amplification method targeting the L452R mutation to detect the Delta variant of SARS-CoV-2

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay’s 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.

Development of a visual detection method of porcine deltacoronavirus using loop-mediated isothermal amplification.

The emergence of porcine deltacoronavirus (PDCoV) presents a significant threat to both human and animal health due to its ability to cause highly contagious enteric diseases. This underscores the crucial need for timely and accurate diagnosis to facilitate effective epidemiological investigation and clinical management. This research aimed to establish a visual detection method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for PDCoV testing. In this study, six pairs of primers were designed according to the conserved sequences of PDCoV ORF1a/b genes. The primer sets and parameters that affect LAMP reaction were optimized. The visual RT-LAMP method was developed by incorporating methyl red into the optimized reaction system, it exclusively detected PDCoV without cross-reactivity with other viruses and the detection limits for PDCoV could reach 10 copies/μL. In comparison with RT-PCR for testing 132 clinical samples, the relative specificity and sensitivity of the visual RT-LAMP were found to be 99.2 and 100%, respectively, with a concordance rate of 99.2% and a kappa value of 0.959, indicating that the visual RT-LAMP is a reliable method for the application of PDCoV detection in clinical samples.

Reverse transcriptase loop-mediated isothermal amplification assisted with localized surface plasmon resonance of gold nanorods for viability assessment of Lactobacillus acidophilus in yogurt

In light of the growing production and consumption of functional foods, it is crucial to develop new methods to assess the authenticity of these products and prevent fraud and mislabeling. In this research, by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the localized surface plasmon resonance (LSPR) effect of gold nanorods (GNRs), the viability of the probiotic bacteria has been evaluated. The RT-LAMP-GNR method effectively amplifies and identifies bacterial 16S-rRNA within 60 min. The efficiency of the RT-LAMP method was assessed by its implementation using RNA isolated from Lactobacillus acidophilus strain (L. acidophilus) and commercial probiotic yogurts. The method's limit of detection (LoD) was 104 bacterial cells per ml, and its sensitivity was 10−5 ng/ml RNA.

Abstract PO5-16-03: RT LAMP assay for the subtyping of breast cancer

Abstract Introduction Our previous study showed that direct reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay can be employed as a substitute in order to detect tumor involvement of lymph nodes within breast cancer patients. In this research, we aimed to investigate the potential applicability of RT-LAMP for the classification of breast cancer subtype. Method Breast Cancer Cell line RNA extraction Total RNA from the cell lines was isolated from cultured cells using RNAiso Plus (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions; MCF10A, MCF7, T47D, BT474, SKBR3, MB468, HCC1806, MB231 Primer design For the Primer design, ER, PR, HER2, Ki 67, CK7 and GATA were targeted. LAMP primers targeted at ER were created between exons 5 and 6, PR were created between exons 6 and 7, HER2 were created between exons 17 and 18, KI67 were created between exons 2 and 3, CK7 were created between exons 5 and 6, and GATA3 were created between exons 2 and 3 utilizing Primer Explorer V4 (http://primereplorer.jp/elamp4.00/index.html). RT-LAMP assays RT-LAMP assays were carried out within a 25 µL reaction which included 12.5 µL of 2 × reaction buffer, 2µL of each primer, 1 µL of enzyme mixture (16U Bst DNA polymerase and 120U M-MLV reverse transcriptase) 8 µL of DEPC-treated water, and 2 µL of the template RNA. DEPC-treated water was also employed for purposes of a negative control. The reaction mixture was conducted within qRT-PCR. Results ER, PR, HER2, Ki-67, CK7, GATA3 expression level were checked for each breast cancer cell line using RT-LAMP. The luminal cell line exhibited elevated levels of ER and PR expression, while the her2 cell line demonstrated a high level of expression for her2. In the RT LAMP assay, there is a direct correlation between shorter detection time and higher expression levels of the substance. Specifically, in the luminal type, ER/PR was detected within a timeframe of 10 to 12 seconds, while HER2 was detected within a timeframe of 15 to 16 seconds. The expression levels of ER, PR, and HER2 were compared between MCF7 (luminal A) and MDA-MB-231 (TNBC) cell lines. RT-LAMP was performed to examine the RNA expression levels, while RT-PCR was applied to confirm the cDNA expression levels. MCF7 and MDA-MB-231 were mixed at 1:9, 5:5 and 9:1 ratios, respectively, and the difference in ER/PR/HE2 expression level was confirmed (Table1, Figure1). The presence of ER was detected in the MCF7 cell line at 14.44 minutes, and PR was observed at 18.84 minutes. HER2 was detected at a similar time (approximately 15 minutes) in both the MCF7 and MB 231 cell lines. Conclusion This study demonstrated that ER, PR, and HER2 can be quantified using RT-LAMP. Ongoing experiments are being conducted to classify breast cancer using the RT-LAMP method, aiming to enhance sensitivity in the quantification of ER, PR, and HER2. Additionally, new primers are being developed specifically for HER2 detection. RT-LAMP offers the advantages of being a point-of-care test that can be conducted on-site and reducing the discrepancies between pathologists. Citation Format: In Hee Lee, Byeongju Kang, Jeeyeon Lee, Jin Hyang Jung, Ho Yong Park, Nora Jee-Young Park, Ji-Young Park, Eun Ae Kim, Jieun Kang, Yee Soo Chae, Soo Jung Lee. RT LAMP assay for the subtyping of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-03.

Comparison of Elva Diagnostic SARS-CoV-2 Saliva Nucleic Acid Test Kit with RT-qPCR and Rapid Antigen Tests in COVID-19 Patients

The practical application of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technique has been confirmed in diagnosing different viral infections. Nevertheless, its accuracy in identifying SARS-CoV-2, particularly in practical clinical situations, has not been thoroughly investigated. This study aims to investigate the sensitivity and specificity of the Elva Diagnostic SARS-CoV-2 Saliva Nucleic Acid Test Kit, utilizing the RT-LAMP and Rapid Antigen Test (RAT) methods for in vitro diagnostic testing, compared to the real-time quantitative polymerase chain reaction (RT-qPCR) method throughout the progression of COVID-19. Method: This study employed an analytical observational diagnostic test at Dr. Soetomo Regional Public Hospital, Surabaya, from March 2022 to May 2022. This research involved 54 samples of saliva and nasopharyngeal swabs obtained from 36 patients confirmed positive for COVID-19 and 18 samples from subjects not confirmed to have COVID-19, tested using the RT-qPCR method. The diagnostic performance of both the RT-LAMP and RAT methods was assessed by calculating their sensitivity and specificity in comparison to RT-qPCR, beginning from the time the patient was confirmed positive for COVID-19. The suitability of each method was analyzed using Cohen’s kappa. The nucleocapsid (N) protein gene from SARS-CoV-2 RNA was detected with RT-LAMP and RAT test kits which showed incompatibilities with the RT-qPCR method (p value 0.308). The positive and negative results with the RT-LAMP and RAT method examinations were similar in number compared to the RT-qPCR method, where the positive results in the RT-LAMP and RAT methods were 2 subjects and the negative results were 52 subjects. Based on the results, only 2 confirmed cases had positive results with RT-LAMP and RAT, which means the sensitivity of both tests is only 5.5% and both are poor screening tests for patients suspected of having COVID-19. In addition, the specificity of RT-qPCR as the gold standard examination method for diagnosing COVID-19 cannot be replaced by the RT-LAMP and RAT methods.

Developing lateral-flow devices for the fast and cheap detection of SARS-cov-2 in wastewater: a potential tool to monitoring local virus outbreaks by wastewater based epidemiology

The SARS-CoV-2 virus generates severe respiratory tract complications such as pneumonia and bronchitis and mild symptoms such as common colds or asymptomatic conditions. The SARS-CoV-2 presence in human feces and in treated/untreated wastewater suggests a transmission way that could generate local outbreaks, in addition to other type of diseases or disorders. Based on the above, in this work it was proposed the assembly of a lateral flow device (LFD) to determine the SARS-CoV-2 presence in wastewater samples. In the LFD a wastewater sample capillary flowed through four membranes: sample zone, conjugate delivery zone, reaction zone and the reactive adsorption zone. The virus amplification was achieved by the novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) at the sampling point. The membranes preconditioning processes and the use of membranes with 5-20 nm porous size increased the capillary flow rate and it was promoted the interaction of the gen of SARS-CoV-2 with the capture agents in the reactive adsorption zone. Additionally, the sensibility of the detection was improved using several methods for the immobilization of the capture agents on the reaction zone membrane. The RT-LAMP method combined with the assembled LFD allowed an efficient SARS-CoV-2 detection at the sampling point in a simple way, cheap and fast compared to conventional and expensive RT-PCR.

Sensitive and Visual Detection of Brassica Yellows Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12 Assay.

Brassica yellows virus (BrYV) is an economically important virus on cruciferous species. In this study, a one-pot reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was developed for the detection of BrYV. The limit of detection of this method reached 32.8 copies of the BrYV ORF5, which is 100-fold more sensitive than the RT-LAMP method. Moreover, there was no cross-reactivity with other rapeseed-infecting RNA viruses or poleroviruses. We dried the CRISPR/Cas12a reagent in a trehalose and pullulan mixture to retain its efficacy at the RT-LAMP temperature of 63°C in order to allow portable BrYV detection in a water bath. The entire process can be performed in about 1 h, and a positive result can be rapidly and conveniently detected using a handheld UV lamp. In the field, the RT-LAMP-CRISPR/Cas12a assay was accurate and had higher sensitivity than RT-LAMP and reverse transcription-polymerase chain reaction assays. The novel RT-LAMP-CRISPR/Cas12a assay allows convenient, portable, rapid, low-cost, highly sensitive, and specific detection of BrYV and has great potential for on-site monitoring of BrYV.

The Performance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Virus Using the Colorimetric Reverse-Transcription Loop Mediated Isothermal Amplification (RT-LAMP) Method in Saliva Specimens of Suspected COVID-19 Patients.

Corona Virus Disease-19 (COVID-19) is a disease caused by Severe-Acute-Respiratory-Syndrome-Coronavirus-2 (SARS-CoV-2). The most reliable and widely accepted method for diagnosing this infection, despite facing various challenges, is the Reverse Transcription Polymerase Chain Reaction (RT-PCR) method, which utilizes nasopharyngeal swab sample. Reverse-transcription loop mediated isothermal amplification (RT-LAMP) is a simpler nucleic acid amplification method compared to the RT-PCR method. This method has several advantages, including: of amplification at constant temperature, faster results, and potentially greater examination capacity. This study aimed to compare the validity of the RT-LAMP method using saliva specimens with that of the RT-PCR method using nasopharyngeal smears. This was an analytical observational study with a cross-sectional design. The participants were inpatients in the COVID-19 special isolation building of Hasan Sadikin General Hospital, Indonesia with a probable (clinical symptoms of covid, but not confirm NAAT examination) or confirmed diagnosis of COVID-19 from September 2021 to February 2022. The inclusion criteria are COVID-19 patients with symptoms, adult subjects, and composite mentions. Patients who were unable to secrete saliva were also excluded. In total, 118 specimens were collected. The validity test results of the saliva specimens using the RT-LAMP method showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), of 65.5%, 100%, 100%, and 75%, respectively. The results increased in subjects treated between 3 and 7 days after symptom onset ie 73.2%, 100%, 100%, and 82.3%, respectively. The very strong specificity accompanied by good sensitivity and NPV in the group of subjects treated 3-7 days after the onset of symptoms indicates that the RT-LAMP method using saliva specimens can be an efficient and reliable alternative tool in detecting the SARS-CoV-2 virus.

Colorimetric RT-LAMP Detection of Multiple SARS-CoV-2 Variants and Lineages of Concern Direct from Nasopharyngeal Swab Samples without RNA Isolation.

Since, during the Coronavirus disease 19 (COVID-19) pandemic, a large part of the human population has become infected, a rapid and simple diagnostic method has been necessary to detect its causative agent, the Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2), and control its spread. Thus, in the present study, we developed a colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) kit that allows the detection of SARS-CoV-2 from nasopharyngeal swab samples without the need for RNA extraction. The kit utilizes three sets of LAMP primers targeting two regions of ORF1ab and one region in the E gene. The results are based on the colorimetric change of hydroxynaphthol blue, which allows visual interpretation without needing an expensive instrument. The kit demonstrated sensitivity to detect between 50 and 100 copies of the viral genome per reaction. The kit was authorized by the National Administration of Drugs, Food and Technology (ANMAT) of Argentina after validation using samples previously analyzed by the gold standard RT-qPCR. The results showed a sensitivity of 90.6% and specificity of 100%, consistent with conventional RT-qPCR. In silico analysis confirmed the recognition of SARS-CoV-2 variants of concern (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427, and B.1.429), and lineages of the Omicron variant (B.1.1.529) with 100% homology. This rapid, simple, and sensitive RT-LAMP method paves the way for a large screening strategy to be carried out at locations lacking sophisticated instrumental and trained staff, as it particularly happens in regional hospitals and medical centers from rural areas.

A Reverse-Transcription Loop-Mediated Isothermal Amplification Technique to Detect Tomato Mottle Mosaic Virus, an Emerging Tobamovirus.

Tomato mottle mosaic virus (ToMMV) is an emerging seed-transmissible tobamovirus that infects tomato and pepper. Since the first report in 2013 in Mexico, ToMMV has spread worldwide, posing a serious threat to the production of both crops. To prevent the spread of this virus, early and accurate detection of infection is required. In this study, we developed a detection method for ToMMV based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP). A LAMP primer set was designed to target the genomic region spanning the movement protein and coat protein genes, which is a highly conserved sequence unique to ToMMV. This RT-LAMP detection method achieved 10-fold higher sensitivity than conventional RT-polymerase chain reaction methods and obtained high specificity without false positives for closely related tobamoviruses or healthy tomato plants. This method can detect ToMMV within 30 min of direct sampling of an infected tomato leaf using a toothpick and therefore does not require RNA purification. Given its high sensitivity, specificity, simplicity, and rapidity, the RT-LAMP method developed in this study is expected to be valuable for point-of-care testing in field surveys and for large-scale testing.

One-Step Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Canna Yellow Streak Virus.

Canna yellow streak virus (CaYSV) is a potyvirus that causes severe damage to the ornamental plant canna in the United Kingdom and Brazil. Here we identified CaYSV in China by isolating total RNA from infected plant, amplifying the virus genome segments, cloning and sequencing the amplicons. After assembly, the full-length genome of the virus was obtained and uploaded to the NCBI database. Phylogenetic analysis results showed that the Guizhou isolate (OL546222) was most closley related to the KS isolate (MG545919.1). Virus detection is essential for virus disease control, but the subclinical infection of CaYSV on canna in its early development increases the difficulty of CaYSV diagnosis. The goal of this study was to develop an efficient method for detection of CaYSV. We designed the primers, optimized the reaction conditions, and finally established a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. The product of RT-LAMP can be analyzed by both agarose gel electrophoresis and visible color change. The established one-step RT-LAMP assay showed high specificity and sensitivity in detecting CaYSV. This RT-LAMP method was also applied in analysis of 61 field samples collected from Guizhou and Jiangsu provinces. The results showed that the infection rates of CaYSV on canna samples from these two provinces were very high (63% and 96% respectively).

A triple-target reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and accurate detection of SARS-CoV-2 virus

The spreading of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across the world has impacted people's health and lives worldwide in recent years. Rapid and accurate diagnosis is crucial for curbing the pandemic of coronavirus disease 2019 (COVID-19). Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has great potential for SARS-CoV-2 detection but fails to completely replace conventional PCR due to the high false-positive rate (FPR). We proposed a triple-target RT-LAMP method for dual-signal, sensitive, and simultaneous detection of conserved genes of SARS-CoV-2. Multiple LAMP primer sets were designed for N, E, and M genes and their amplification efficacy were screened. Then, using artificial plasmids and RNA, the optimal primer set for each gene was examined on specificity, sensitivity, and detection range. The RT-LAMP initiated by these primer sets exhibited better specificity and sensitivity than that of RT-qPCR, and the triple-target RT-LAMP could determine different variants of SARS-CoV-2. By testing 78 artificial RNA samples, the total FPR of triple-target RT-LAMP was eliminated compared with that of mono-target RT-LAMP. The triple-target RT-LAMP method precisely identified throat swab specimens through colorimetry and fluorescent signals within 60 min, and the limit of detection (LOD) was as low as 187 copies/reaction. In the future, the triple-target RT-LAMP can be applied to in-field and on-site diagnosis of symptomatic and asymptomatic virus carriers.

RT-LAMP Multicenter Study for SARS-CoV-2 Genome Molecular Detection in Brazilian Swab and Saliva Samples.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid method that can replace RT-qPCR. A simple molecular assay for SARS-CoV-2 RNA detection in gold-standard diagnosis through swabs and alternative specimens such as saliva could be helpful in promoting genomic surveillance. A multicenter study was conducted to evaluate the RT-LAMP assay method as an alternative for the molecular detection of SARS-CoV-2 lineages in swab and saliva samples. A total of 350 swabs from individuals with (n = 276) or without (n = 74) COVID-19 tested by RT-qPCR were collected. Paired saliva was also collected from 90 individuals who had SARS-CoV-2 RNA that was detectable (n = 30) or undetectable (n = 60) via RT-qPCR. For the RT-LAMP methodology, six primers were used for ORF1 gene amplification. As for SARS-CoV-2 genotyping, 39 swabs had the whole genome sequenced by MinION. The sensitivity of RT-LAMP to the swab was 90.2%. For the swab samples with Ct ≤ 30, the sensitivity improved by 96%. Considering saliva with Ct ≤ 30 in RT-qPCR testing, the RT-LAMP sensitivity was 100%. The RT-LAMP specificity was 100% for both the swab and saliva samples. This RT-LAMP assay was capable of detecting all the SARS-CoV-2 lineages circulating in the Brazilian swab samples. The RT-LAMP method has significant potential for use in clinical routines since it was capable of detecting SARS-CoV-2 RNA in swab and saliva samples.

Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method.

Simple SummaryPorcine epidemic diarrhea (PED) is a severe disease which has led to tremendous economic losses in the swine industry all over the world. The early detection of its pathogen (PEDV) is vital to prevent and cure this disease. Here, we report the development of a new visual diagnostic test for PEDV—the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. This new assay proved to be specific and sensitive when applied to clinical specimens.Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 °C and 3 min at 80 °C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/μL. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis.

Rapid and Reliable Detection of SARS-CoV-2 Using Direct RT-LAMP.

Background: The global pandemic coronavirus SARS-CoV-2 has a healthcare, social and economic burden. To limit the spread of the virus, the World Health Organization (WHO) urgently called for extensive screening of suspected individuals; thus, a quick, simple, and sensitive diagnostic assay is always in need. Methods: We applied reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of SARS-CoV-2. The RT-LAMP method was optimized by evaluating two fluorescence amplification mixes and several reaction times, and results were compared to the standard real-time RT-PCR (rtRT-PCR). The assay was validated using 200 nasopharyngeal swabs collected in viral transport media (62 positive for SARS-CoV-2, and 138 negative for SARS-CoV-2 detected by the rtRT-PCR method). The samples were diluted 1:4 in diethylpyrocarbonate (DEPC)-treated water, utilized for RT-LAMP using different singleplex and multiplex sets of LAMP primers (N gene, S gene, and orf1ab gene), and incubated at 65 °C using real-time PCR 7500. Results: Our direct detection with the RT-LAMP protocol showed 100% concordance (sensitivity and specificity) with the standard protocol used for the detection of SARS-CoV-2 nucleic acid. Conclusions: In this study, we set up a rapid, simple, and sensitive RT-LAMP assay for the detection of SARS-CoV-2 in clinical samples. The assay is suitable for point of care detection in public hospitals, medical centers in rural areas, and in transportation hubs.

Rapid detection of Potato leafroll virus and Potato virus Y by reverse transcription loop-mediated isothermal amplification method in north-east India

Potato leafroll virus (PLRV) and Potato virus Y (PVY) are two important viruses causing serious potato yield losses in the North-east region and other planting areas in India. As a consequence, it is urgent to develop an efficient and quick method for the identification and diagnosis in the field. The results presented here showed that the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was efficient and sensitive than reverse transcription-polymerase chain reaction (RT-PCR) for the detection of PLRV and PVY. The RT-LAMP primers specifically targeted PLRV and PVY (including PVYO, PVYN, and PVYNTN strains) and resulted in typical sigmoidal amplification curves. Ten-fold serial dilutions of PLRV and PVY total RNA indicated that RT-LAMP is faster and at least a hundred times more sensitive than RT-PCR in detecting both the viruses. Additionally, samples that RT-PCR could not detect at a diluted concentration of 10−3 and 10-4 ng/μl were identified by RT-LAMP. Thus, RT-LAMP offers many advantages over RT-PCR such as low cost and high accuracy, sensitivity, and specificity for the rapid diagnosis of plant virus diseases. In conclusion, the results highlighted the efficacy of the RT-LAMP method in quickly detecting PLRV and PVY in infected plants.

Virus-Like Particles as Positive Controls for COVID-19 RT-LAMP Diagnostic Assays.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and inexpensive isothermal alternative to the current gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, unlike RT-qPCR, there are no consensus detection regions or optimal RT-LAMP methods, and most protocols do not include internal controls to ensure reliability. Naked RNAs, plasmids, or even RNA from infectious COVID-19 patients have been used as external positive controls for RT-LAMP assays, but such reagents lack the stability required for full-process control. To overcome the lack of proper internal and external positive controls and the instability of the detection RNA, we developed virus-like particles (VLPs) using bacteriophage Qβ and plant virus cowpea chlorotic mottle virus (CCMV) for the encapsidation of target RNA, namely a so-called SARS-CoV-2 LAMP detection module (SLDM). The target RNA is a truncated segment of the SARS-CoV-2 nucleocapsid (N) gene and human RNase P gene (internal control) as positive controls for RT-qPCR and RT-LAMP. Target RNAs stably encapsidated in Qβ and CCMV VLPs were previously shown to function as full-process controls in RT-qPCR assays, and here we show that SLDMs can fulfill the same function for RT-LAMP and swab-to-test (direct RT-LAMP with heat lysis) assays. The SLDM was validated in a clinical setting, highlighting the promise of VLPs as positive controls for molecular assays.

Direct Detection of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Without RNA Purification

Coronavirus disease 2019 (COVID-19) is an acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which has spread globally. Shortage of nucleic acid extraction kits may delay the speed of diagnosis. In order to address this need, we propose a simple method for direct RNA extraction from nasopharyngeal swab samples without RNA purification. Here, we describe a reverse transcription loop-mediated isothermal amplification technique with 95.4% sensitivity (95% CI: 84.2-99.4%) and 100% specificity (95% CI: 78-100%). Combined with a simple RNA extraction step, this optimized RT-LAMP method is able to amplify SARS-CoV-2 directly from clinical nasopharyngeal swab samples in viral transport media in 50 minutes. This method provides an opportunity to facilitate SARS-CoV-2 detection especially in resource limited settings where nucleic acid extraction kits are few.Funding: This study was supported by Prototype Research Grant Scheme (PRGS), PR001-2020B (PRGS/2/2020/SKK09/UM/02/1) from the Ministry of Education, Malaysia.Declaration of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.Ethics Approval Statement: Ethical approvals for this study was obtained from UMMC 85 Medical Ethics Committee (202041-8418) and Medical Research Ethics Committee 86 (MREC) Ministry of Health Malaysia (NMRR-20-2344-56994).

Detection and discrimination of multiple strains of Zika virus by reverse transcription-loop-mediated isothermal amplification

BackgroundMonitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries.ResultsCommon primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10−2 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present.ConclusionsThe RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.

Detecting SARS-CoV-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (LAMP) to polymerase chain reaction (PCR)

BackgroundA cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting.MethodsThis prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines.ResultsThe novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19.ConclusionsRT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.