Metodo LAMP como alternativa diagnostica para la deteccion del virus SARS-CoV-2

Summary Introduction: The SARS-CoV-2 coronavirus, that causes the COVID-19 disease, has become a global public health problem that requires the implementation of rapid and sensitive diagnostic tests. Aim(s): To evaluate and compare the sensitivity of LAMP assay to a standard method and use RT-LAMP for the diagnosis of SARS-CoV-2 in clinical samples from Colombian patients. Method(s): A descriptive and cross-sectional study was conducted. A total of 25 nasopharyngeal swab samples including negative and positive samples for SARS-CoV-2 were analyzed, through the RT-LAMP method compared to the RT-qPCR assay. Result(s): LAMP method detected ~18 copies of the N gene, in 30 min, evidenced a detection limit similar to the standard method, in a shorter time and a concordance in RT-LAMP of 100% with the results. Conclusion(s): RT-LAMP is a sensitive, specific, and rapid method that can be used as a diagnostic aid of COVID-19 disease.Copyright © 2021. All Rights Reserved.

Comparison on the performance of two qPCR instruments in the detection of SARS-CoV-2 virus

Objective To compare the performance of two qPCR instruments in detecting SARS-CoV-2 virus in the nasopharyngeal swab samples of suspected COVID-19 isolated individuals in Jinghu district Wuhu city.Methods A total of 151 nasopharyngeal swab samples were collected from individuals with suspected COVID-19isolated during January 2021 and July 2022 at a quarantine site in the Jinghu district. Nucleic acid of SARS-CoV-2virus was quantified parallelly using ABIQ5 real-time fluorescence quantitative analyzer（Q5 analyzer） and Bole CFX96 fluorescence quantitative PCR analyzer（Bole analyzer） in the laboratory. Q5 analyzer was used as the reference instrument, while Bole analyzer was used as an experimental instrument. The detection results of N gene, ORF1ab fragment and CT value of the two RT-PCR machines were analyzed and compared using paired four grid test, Spearman test and paired sample t-test in SPSS 22 statistical software. Results The results of 151samples for different target genes tested by two instruments were in good agreement（N gene: Kappa=1, P＜0. 05;ORF1ab fragment: Kappa=0. 972, P＜0. 05）. The inter-batch repeatability rates were 4. 01% and 3. 04%for N gene and ORF fragment of the same batch positive quality controls by Q5 analyzer, and were 4. 90% and 3. 57% by Bole analyzer. The intra batch repeatability rates of the two instruments at different hole locations were similar, and CV values were less than 3%. The results of 23 positive samples showed that the differences in CT values of N gene（29. 38±7. 22） and ORF1ab（30. 83±6. 27） detected by Q5 analyzer were statistically significant（t=2. 765, P＜0. 05）, while the differences in CT values of N gene（29. 58±7. 27） and ORF1ab（30. 77±8. 02） detected by Bole analyzer were not statistically significant（t=1. 753, P＞0. 05）. The correlation coefficients of CT values of different target genes detected by the two instruments were rN=0. 960 and rORF=0. 865, showing correlated CT values（P＜0. 05）. Conclusion The CT values of N gene and ORF1ab fragment of SARS-CoV-2 virus detected by the two instruments have strong correlation and agreement, indicating that either of the instrument can be used for laboratory sample detection and analysis. The repeatability of Q5 analyzer is better than that of Bole analyzer. The detection stability of ORF fragments of both instruments is better than that of N gene, and the detection sensitivity of Q5 analyzer for N gene is higher than that for ORF fragment. The sample tubes should be placed in the middle of the PCR machine in order to reduce the system error.

Performance Evaluation of SARS-CoV-2 Viral Transport Medium Produced by Bangladesh Reference Institute for Chemical Measurements.

A viral transport medium (VTM) was developed following the Centers for Disease Control and Prevention, USA (US-CDC) standard operating procedure (SOP) DSR-052-05 with necessary improvisation and was used for storing coronavirus disease 2019 (COVID-19) swab specimens. Considering Bangladesh's supply chain and storage conditions, improvisation was essential for extending sample storage time while retaining efficiency. In-house VTM was produced using Hank's balanced salt solution (HBSS) supplemented with 1% bovine serum albumin V (BSA), 0.5 µg /mL of gentamicin sulfate, and 100 µg/mL of fluconazole. The produced VTM composition, quality, sterility, specificity, and efficiency were verified in-house and through an independent contract research organization (CRO). An accelerated stability study projected that under the recommended temperature (4 °C), it would remain stable for four months and preserve samples for over a month. The real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) test detected the targeted N gene and ORF1ab gene from the VTM stored samples. Our VTM is equally as effective as the Sansure Biotech VTM in keeping SARS-CoV-2 RNA specimens detectable in rRT-PCR (100% sensitivity and specificity in random and blinded samples). In conclusion, the BRiCM VTM will make the battle against pandemics easier by effectively collecting and storing nasopharyngeal and oropharyngeal swabs for COVID-19 detection.

'CoviSwiftTM': A point-of-care RT-PCR device for SARS-CoV-2 and its variant detection.

We present the point-of-care (POC) molecular diagnostic solution, evaluated during COVID-19 pandemic caused by SARS-CoV-2 (Dec 2021). The POC comprised of a complete platform from self-sampling to RT-PCR testing of SARS-CoV-2 and its variants on portable Compact Q Real time polymerase chain reaction system. The multiplex assay was designed to target S, ORF1, and N genes of SARS-CoV-2 genome in a single tube with RNaseP as endogenous internal control. The present POC enables high accuracy (>95%) and high-throughput testing with a turnaround time of 45 min. It provides a unique platform from self-sample collection to report generation with rapid protocol, pipette and expert-free operation, long shelf-life stability and room temperature storage which enable to increase the efficiency of molecular testing. This novel test named "CoviSwift™ COVID-19 S PLUS RAPID PCR KIT" has been approved by CDSCO, Indian National Regulatory Authority, India, and is in use for clinical settings in India.

A Combined Method Based on the FIPV N Monoclonal Antibody Immunofluorescence Assay and RT-nPCR Method for the Rapid Diagnosis of FIP-Suspected Ascites

Feline infectious peritonitis (FIP), which is caused by feline infectious peritonitis virus (FIPV), is a fatal and immunologically mediated infectious disease among cats. At present, due to the atypical clinical symptoms and clinicopathological changes, the clinical diagnosis of FIP is still difficult. The gold standard method for the differential diagnosis of FIP is immunohistochemistry (IHC) which is time-consuming and requires specialized personnel and equipment. Therefore, a rapid and accurate clinical diagnostic method for FIPV infection is still urgently needed. In this study, based on the etiological investigation of FIPV in parts of southern China, we attempted to explore a new rapid and highly sensitive method for clinical diagnosis. The results of the etiological investigation showed that the N gene of the FIPV BS8 strain had the highest homology with other strains. Based on this, a specific FIPV BS8 N protein monoclonal antibody was successfully prepared by expression of the recombinant proteins, immunization of mice, fusion and selection of hybridoma cell lines, and screening and purification of monoclonal antibodies. Furthermore, we carried out a time-saving combination method including indirect immunofluorescence assay (IFA) and nested reverse transcription polymerase chain reaction (RT-nPCR) to examine FIP-suspected clinical samples. These results were 100% consistent with IHC. The results revealed that the combined method could be a rapid and accurate application in the diagnosis of suspected FIPV infection within 24 hours. In conclusion, the combination of IFA and RT-nPCR was shown to be a fast and reliable method for clinical FIPV diagnosis. This study will provide insight into the exploitation of FIPV N antibodies for the clinical diagnosis of FIP-suspected ascites samples.

Challenges of Singapore's First Acute Geriatric Isolation Facility During the COVID-19 Pandemic

Aim: The COVID-19 pandemic has seen hospitals isolating suspect cases. Geriatric populations are at a risk of severe COVID-19 disease and often present with concomitant geriatric syndromes requiring holistic interdisciplinary care. However, isolation of older people poses challenges to care delivery. This study shares the experience of Singapore's first acute geriatric isolation facility geriatric PARI (Pneumonia-Acute Respiratory Infection) ward and describes the geriatric-related outcomes and pitfalls in care delivery. Method(s): This is a retrospective cross-sectional study performed in 7 negative pressure isolation rooms in an acute care public hospital in Singapore. 100 patients admitted consecutively to the geriatric PARI ward were included. Patient demographics, presenting symptoms and geriatric-related adverse outcomes associated with hospitalisation were collected and analysed. Result(s): Patients' mean age was 86.4 years (standard deviation [SD]: 6.8) with significant comorbidities being hypertension (81%), hyperlipidaemia (74%) and renal disease (70%). 51% of patients had dementia and 24% had behaviour and psychological symptoms of dementia (BPSD). 27% of patients presented atypically with delirium and 15% presented with a fall. Delirium was associated with restraint use (OR: 3.88;p-value 0.01). Falls rate was 1.64 per 1000 occupied bed. 1 patient screened positive for COVID-19. Conclusion(s): The geriatric PARI ward is essential for curbing nosocomial transmission of COVID-19. This is important in the older people with comorbidities who are more likely to develop morbidity and mortality. Our study reveals challenges in delivering person-centred care to the older patients in isolation rooms, especially in the management of delirium and falls prevention. Innovative strategies should be developed to minimise isolation-related adverse outcome.Copyright © The Author(s) 2022.

Diagnosing COVID-19: diagnostic importance of detecting E gene of the SARS-CoV-2 genome

Aim: To evaluate the significance of E gene analysis in addition to N and RdRp genes of SARS-CoV-2, and to compare the specificity and sensitivity of targets. Material(s) and Method(s): We used two reverse transcription-PCR assays: one targeting N, E and RdRp and the other targeting N and RdRp genes and analyzed variation in threshold cycle (Ct) values. Result(s): Of the 155 samples, 70.32% tested positive: all three genes were detected in 45.87%, N and RdRp in 19.27% and only N in 34.86%. Patients negative for the E gene were tested after symptoms disappeared and Ct values were significantly higher. Conclusion(s): Samples negative for the E gene were potentially false positive and clinical conditions should be assessed while interpreting results.Copyright © 2023 Future Medicine Ltd.

Detection and molecular characterization of gammacoronavirus in quail population in Iran

BACKGROUND: Gammacoronaviruses, which are single-stranded, positive-sense RNA viruses, are responsible for a wide variety of existing and emerging diseases in birds. The Gammacoronaviruses primarily infect avian hosts. OBJECTIVE(S): This study aimed to investigate the genetic diversity of Gammacoronaviruses in quail population in Iran. METHOD(S): In the period from 2016 to 2018, samples from 47 quail flocks with or without enteric signs, were collected from four provinces in Iran. RESULT(S): Gammacoronavirus was detected in samples of 4 flocks by using RT-PCR and characterized by N gene sequencing. The isolates formed a distinct group from other Gamma- coronaviruses groups CONCLUSION(S): The finding suggests the existence of a novel Gammacoronavirus circulating in quail farms. The phylogenetic relationship of the isolates concerning different sequences and geographical regions displayed complexity and diversity. The present study is the first detection of Gammacoronavirus in quail farms in Iran. Further studies are required and should include the isolation and experimental studies of Gammacoronaviruses in Iran.Copyright © 2019.

Stealth Omicron: A Novel SARS-CoV-2 Variant That Is Insensitive to RT-qPCR Using the N1 and N2 Primer-Probes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is causing a worldwide pandemic since the spring of 2020. In Osaka, the second biggest prefecture in Japan, we identified a novel SARS-CoV-2 variant from a coronavirus disease 2019 (COVID-19) patient that was detected by polymerase chain reaction (PCR) using E primers, but not by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) using the N1 and N2 primer-probe sets recommended by CDC. We analyzed the S and N gene regions by reverse-transcription and nested PCR using the S and N specific primers, and DNA sequencing, and found that this BA.5 variant contained point mutations in the probe sequences of both the N1 and N2 primer-probe regions. This finding led us to affirm the importance of monitoring the genome sequence of the SARS-CoV-2 variants continuously.

Photoactive conjugated microporous polymer/carbon nanotube coupled with T-junction recycling dual-strand displacement amplification for sensing N-Gene of COVID-19

In this work, a novel conjugated microporous polymer on carbon nanotube composite (CMP-CNTs) was synthesized as photoelectrochemical (PEC) signal probe to construct sensitive PEC biosensor for sensing N-Gene of COVID-19 by integrating with an ingenious target-trigger T-junction recycling dual-strand displacement amplification (T-DSDA). The CMP-CNTs composites has an ideal photoelectrical conversion efficiency owing to the appearance of a good band matching that can effectively promote electron-hole pairs separation and accelerate carrier migration, thereby generating an extremely high initial photocurrent. Meanwhile, the T-DSDA with superior target conversion efficiency to traditional approaches could convert the small number of targets into extensive output DNAs, leading to the in-situ generation of quench agent N-GQDS decorated nanowires on electrode for significantly reducing initial photocurrent. The results demonstrated that proposed PEC biosensor had a high sensitivity towards N-Gene of COVID-19 and the detection limit was 33 aM, which provided a new way to build the simple, sensitive, and reliable sensing platform for great potential in biological analysis and early clinical diagnosis. [Display omitted] • New CMP-CNTs composites have a good energy band matching, which can boost visible light absorption efficiency. • A target-trigger T-junction dual-strand displacement amplification transform small targets into massive output DNAs. • Output DNAs trigger HCR to generate N-GQDs modified nanowires in situ, which significantly reduces the PEC signal. • The proposed PEC biosensor of CMP-CNTs composites achieved ultra-sensitive detection of N-Gene of COVID-19. [ FROM AUTHOR]

Tailored Multiplex Real-Time RT-PCR with Species-Specific Internal Positive Controls for Detecting SARS-CoV-2 in Canine and Feline Clinical Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.

Establishment and evaluation of fluorescent RT-RAA assay for SARS-CoV-2 sgRNAs detection

Objective To establish a reverse-transcription recombinase-aided amplification assay（RT-RAA） to rapidly detect SARS-CoV-2 sub-genomic RNAs（sgRNAs）. Methods The primers and probe for isothermal nucleic acid amplification were designed based on the 5′-leader and 7a and N gene sequence of SARS-CoV-2, and the sgRNAs of SARS-COV-2 were rapidly detected within 30 min at 39 ℃.The sensitivity, specificity and consistency of the assay were evaluated. Results The detection limit of the method was 20 copies/μl and there were no cross-reactions with other respiratory pathogens, showing decent sensitivity and specificity.The results of the assay were concordant with that of real-time PCR, indicating a better consistency of two methods（κ=0.762,P<0.001）. Conclusions The fluorescence RT-RAA assay established in the study can be used for the rapid detection of SARS-CoV-2 sgRNAs, which is of great significance for the rapid diagnosis of COVID 19.

Photoactive Conjugated Microporous Polymer/Carbon Nanotube Coupled with T-junction Recycling Dual-Strand Displacement Amplification for Sensing N-Gene of COVID-19

In this work, a novel conjugated microporous polymer on carbon nanotube composite (CMP-CNTs) was synthesized as photoelectrochemical (PEC) signal probe to construct sensitive PEC biosensor for sensing N-Gene of COVID-19 by integrating with an ingenious target-trigger T-junction recycling dual-strand displacement amplification (T-DSDA). The CMP-CNTs composites has an ideal photoelectrical conversion efficiency owing to the appearance of a good band matching that can effectively promote electron-hole pairs separation and accelerate carrier migration, thereby generating an extremely high initial photocurrent. Meanwhile, the T-DSDA with superior target conversion efficiency to traditional approaches could convert the small number of targets into extensive output DNAs, leading to the in-situ generation of quench agent N-GQDS decorated nanowires on electrode for significantly reducing initial photocurrent. The results demonstrated that proposed PEC biosensor had a high sensitivity towards N-Gene of COVID-19 and the detection limit was 33 aM, which provided a new way to build the simple, sensitive, and reliable sensing platform for great potential in biological analysis and early clinical diagnosis.

Characterisation of ORF3, M, N and E Gene Sequences of Porcine Epidemic Diarrhoea Virus from Domestic Pigs in Poland.

Introduction: Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen causing porcine epidemic diarrhoea and acute gastroenteritis in pigs of all ages. Previous analysis of the viral genome of PEDV in Poland was only based on the spike protein (S) gene sequences and no analysis of other genes has been performed. The aim of this study was to analyse the envelope (E), membrane (M) and nucleocapsid (N) protein and open reading frame 3 (ORF3) gene sequences. Material and Methods: Viral RNA from 18 Polish pig faecal samples that were quantitative reverse transcription PCR-positive for PEDV was analysed in four genomic regions (E, M, N and ORF3). Results: Phylogenetic analysis based on these regions' sequences revealed that Polish PEDV isolates were highly related and were clustered into group G2a across the four genes compared. Moreover, the Polish strains were located in distinct subclusters on the phylogenetic trees, which suggests the presence of at least three independently evolving PEDV genetic lines circulating in Poland. The occurrence of unique mutations in the sequences of Polish PEDV strains suggests that PEDV continues to undergo evolutionary processes, accumulating the mutations necessary for viral fitness in its natural hosts. The Polish PEDV strains differed genetically from the CV777 vaccine strain, suggesting the risk of relatively low vaccine efficacy if this strain is used. Conclusion: Our results promote a better understanding of the genetic diversity of PEDV field isolates in Poland and highlight the importance of molecular characterisation of PEDV field strains for the development of an effective vaccine against PEDV.

Determinative Role of Non-structural Protein Genes in Genus Identification of Coronaviruses

Interrelationship of coronavirus genus with key fragments of viral genome was investigated. Genes of structural proteins (S-gene of spike protein and N-gene of nucleocapsid protein) and ORF1ab of polyprotein pp1ab, that in infected cell is split into 16 non-structural proteins, were considered as such fragments. Statistical method based on averaged codon distribution in the genes of genus prototype variants was applied in the work to recognize genus of coronavirus. High reliability of this method has been demonstrated earlier in recognizing the 15 species and serotypes of the flaviviruses, such as viruses of yellow fever, dengue fever, various encephalitides, etc. For each key fragment of the coronavirus genome the numerical experiments on identification of genus for the 3242 viral genomes from the GenBank have been done. The highest reliability (98%) was achieved, when ORF1ab frequency codon characteristics were used. It appeared to be that in recognizing genus of Gammacoronavirus, basing on spike protein gene, about half of the 345 genomes of this genus were identified as Betacoronavirus (84.6%) and Alphacoronavirus (15.4%). Analogous phenomenon of significant error appeared in determinating Alphacoronavirus genus, basing on nucleocapsid protein gene, also. However, these significant errors may be a consequence of the coronavirus genome plasticity in the result of homologous recombinations between the viral genomes. © 2022 IEEE.

Development and Validation of Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple and Rapid Diagnostic Tool for SARS-CoV-2 Detection.

Since the COVID-19 pandemic outbreak in the world, many countries have searched for quick diagnostic tools to detect the virus. There are many ways to design diagnostic assays; however, each may have its limitations. A quick, sensitive, specific, and simple approach is essential for highly rapidly transmitted infections, such as SARS-CoV-2. This study aimed to develop a rapid and cost-effective diagnostic tool using a one-step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) approach. The results were observed using the naked eye within 30-60 min using turbidity or colorimetric analysis. The sensitivity, specificity, and lowest limit of detection (LoD) for SARS-CoV-2 RNA against the RT-LAMP assay were assessed. This assay was also verified and validated against commercial quantitative RT-PCR used by health authorities in Saudi Arabia. Furthermore, a quick and direct sampling from the saliva, or buccal cavity, was applied after simple modification, using proteinase K and heating at 98 °C for 5 min to avoid routine RNA extraction. This rapid single-tube diagnostic tool detected COVID-19 with an accuracy rate of 95% for both genes (ORF1a and N) and an LoD for the ORF1a and N genes as 39 and 25 copies/reaction, respectively. It can be potentially used as a high-throughput national screening for different respiratory-based infections within the Middle East region, such as the MERS virus or major zoonotic pathogens such as Mycobacterium paratuberculosis and Brucella spp., particularly in remote and rural areas where lab equipment is limited.

CRISPR/Cas12a system responsive DNA hydrogel for label-free detection of non-glucose targets with a portable personal glucose meter.

In this work, personal glucose meter (PGM) as a portable electrochemical device was utilized for sensitive detection of non-glucose targets: N-gene and PCB77, respectively. DNA hydrogel, which can respond to CRISPR/Cas system, was prepared for label-free encapsulating invertase. In the presence of targets, the repeated sequence for the activation of Cas12a was obtained due to the performance of RCA. Unlike "one-to-one" recognition, activated Cas12a can efficiently cleave multiple single-stranded linker DNAs on DNA hydrogels, thus releasing many invertase that can be used for PGM detection. With the amplification of RCA and CRISPR/Cas system, high detection sensitivity can be obtained even using portable PGM. The detection limits for N-gene and PCB77 were 2.6 fM and 3.2 × 10-5 µg/L, respectively, with high specificity and good practical application performance. The developed biosensor can be used for online monitoring with the merit of low cost, easy operation and can be used for various targets analysis.

Performance evaluation of antigen detection rapid diagnostic test (Ag-RDT) for COVID-19 diagnosis in a primary healthcare center during the Shanghai COVID-19 quarantine period.

BACKGROUND: Rapid and accurate detection of SARS-CoV-2 infection is the cornerstone of prompt patient care. However, the reliability of the antigen rapid diagnostic test (Ag-RDT) in the diagnosis of SARS-CoV-2 infection remains inconclusive. METHODS: We conducted a field evaluation of Ag-RDT performance during the Shanghai Coronavirus disease 2019 (COVID-19) quarantine and screened 7225 individuals visiting our Emergency Department. 83 asymptomatic SARS-CoV-2 (+) individuals were enrolled in the current study. Simultaneously, Ag-RDT was performed to evaluate its testing performance. RESULTS: For the Ag-RDT(-) cases, the average cycle threshold (Ct) values of the N gene were 27.26 ± 4.59, which were significantly higher than the Ct value (21.9 ± 4.73) of the Ag-RDT(+) individuals (p < 0.0001). The overall sensitivity of Ag-RDT versus that of RT-PCR was 43.37%. The Ag-RDT(+) individuals regarding the N gene's Ct value were 16 cases in the < 20 range, 12 in 20-25, 5 in 25-30, and 3 in 30-35. The corresponding sensitivity was 84.21%, 52.17%, 21.74% and 16.67%, respectively. Meanwhile, sampling had a straight specificity of 100% regardless of the Ct value. CONCLUSIONS: The Ag-RDT were extremely sensitive in asymptomatic COVID-19 individuals with a Ct value < 20.

Potential of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple Rapid Molecular Diagnostic Tool to Detect SARS-CoV-2 Nucleocapsid Gene

Introduction: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), identified in December of 2019, is the cause of the coronavirus disease 2019 (COVID-19). Due to the high reproductive rate of the virus, the best way to slow down the spread is to identify and isolate patients at the early stage of infections. The current diagnostic methods are either too expensive, slow or have low accuracy. Variants of SARS-CoV-2 with mutations at the primer binding sites may cause evasion of polymerase chain reaction (PCR) detection using current primers. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has potential as a rapid molecular test that is easy to conduct. Methods: LAMP primers were design based on the highly conserved regions of the SARS-CoV-2 Nucleocapsid (N) gene. RT-LAMP assays were conducted using an optimized Bst 3.0 polymerase protocol on T7 RNA polymerase synthesized RNA template. The LAMP sensitivity assay was tested on 1:10 serial diluted pJET1.2 vector with SARSCoV2 N gene inserts. A specificity test was conducted by running the test on plasmids containing SARS-CoV and MERS-CoV N genes. The results were visualised via gel electrophoresis, SYBR Green staining and Lateral Flow Dipstick (LFD). Results: The optimized protocol is sensitive enough to detect SARS-CoV-2 genetic material within 10 minutes but is most sensitive at 30 minutes. Additionally, it is specific to only the genetic materials of SARS-CoV-2. Furthermore, an LFD with multiple test lines was successful for multiplexed LAMP reactions with different genic regions of the virus. Conclusion: The multiplexed LFD-LAMP is potentially a simple yet specific and sensitive method of rapid molecular diagnostics of COVID-19.