Accuracy of a raw saliva-based COVID-19 RT-LAMP diagnostic assay

ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1-99.8) with 90.5% specificity (95% CI 69.6-98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7-94.8) and a specificity of 90.9% (95% CI 70.8-98.8). As a result, the accuracy of 92.9% (95% CI 80.5-98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6-94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

Application and Modification of RT-LAMP for Rapid Detection of SARS-CoV-2 Viral Genome

@#COVID-19 outbreak caused by the newly discovered SARS-CoV-2 has become a major public health threat around the world and has create a tremendous effect on the global economy. Hence, there is a high demand for rapid and accurate diagnosis to contain the spread of the disease. The Reverse-Transcription Polymerase Chain Reaction (RTPCR), the current standard for diagnosis of COVID-19 however possesses certain drawbacks that limits its application to meet the high demand of the continually increasing COVID-19 cases. Conversely, Loop-Mediated Isothermal Amplification (LAMP) is another nucleic acid amplification method that shows a great potential as an alternative tool in rapid diagnosis of COVID-19 due to its simplicity and rapidity. This review summarized the recent published research articles related to the application and modification of RT-LAMP assay for the rapid detection of COVID-19 in comparison with other available diagnostic methods.

Evidence of dengue virus in eviscerated specimens of panophthalmitis secondary to dengue fever: A possible cause-effect phenomenon

Purpose: To report a retrospective series of three cases of infectious panophthalmitis post?dengue fever with ex vivo confirmation of dengue virus ribonucleic acid (RNA) in the tissues of the eye. Methods: Four eyes of three patients, who were diagnosed with panophthalmitis following dengue fever and who underwent evisceration, were included. All demographic and clinical data were recorded. The eviscerated samples were subjected to direct microscopy, culture for bacteria, fungi, and parasites, and molecular virology (dengue virus [DENV] NS1?specific reverse transcription loop?mediated isothermal amplification (RT?LAMP) assay). Results: The time from the development of dengue fever to the occurrence of ocular symptoms was 4.33 ± 1.15 (median 5) days. DENV NS1 RNA, suggestive of the presence of the dengue virus, was confirmed in all evisceration specimens (uveal tissue, cornea). All the patients recovered completely from dengue fever and on follow?up had healthy eviscerated sockets. Conclusion: Demonstration of the DENV RNA in the eviscerated specimens of panophthalmitis following dengue fever implicates the DENV in the pathophysiology of the ocular infection.

Development of a RT-LAMP assay for detection of SARS-CoV-2

Background & objectives: The pandemic of SARS-COV-2 began in Wuhan, China in December 2019 and has caused more than 101 million cases worldwide. Diagnostic technologies possessing sensitivity and specificity equivalent to real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assays are needed to ramp up testing capacity in most countries. Newer platforms need to be technically less demanding, require minimum equipment and reduce turn-around time for reporting results. The objective of this study was to exploit loop-mediated isothermal amplification (LAMP) for the detection of SARS-CoV-2 and evaluate its performance by comparison with rRT-PCR. Methods: Reverse-transcription LAMP (RT-LAMP) assay primers were designed to detect envelop (E) and nucleocapsid (N) genes of SARS-CoV-2. Positive control RNA was prepared by in vitro transcription of E and N genes clones. RT-LAMP amplification reactions were incubated at 65°C for 30 min. Results were recorded visually. RT-LAMP results were evaluated by comparing the results obtained with a commercial rRT-PCR kit. Results: The RT-LAMP assay for E and N genes was carried out in separate tubes. RT-LAMP detected about 40 copies of SARS-CoV-2 RNA per reaction. A total of 253 throat swabs were tested using the RT-LAMP assay. The overall diagnostic sensitivity and specificity of the LAMP assay were 98.46 and 100 per cent, respectively, as compared to the rRT-PCR. Interpretation & conclusions: SARS-CoV-2 RT-LAMP assay was designed, standardized and evaluated. The assay showed diagnostic sensitivity and specificity equivalent to rRT-PCR assays. The assay will be useful to increase testing capacity for the detection of SARS-CoV-2 in the country.

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

BACKGROUND Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis. OBJECTIVES To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages. METHODS SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E. FINDINGS SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL. MAIN CONCLUSIONS Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

Método LAMP como alternativa diagnostica para la detección del virus SARS-CoV-2 / LAMP method as a diagnostic alternative for the detection of the SARS-CoV-2 virus / Método LAMP como alternativa de diagnóstico para a detecção do vírus SARS-CoV-2

RESUMEN Introducción: el coronavirus SARS-CoV-2, causante de la enfermedad COVID-19 se ha convertido en un problema de salud pública mundial que requiere la implemen-tación de pruebas de diagnóstico rápidas y sensibles. Objetivo: evaluar y comparar el límite de detección del método LAMP con respecto al método estándar y aplicar el método RT-LAMP para el diagnóstico de SARS-CoV-2 en muestras clínicas de pacientes colombianos. Métodos: se realizó un estudio descriptivo y transversal, analizando un total de 25 muestras de hisopado nasofaríngeo incluyendo muestras negativas y positivas para SARS-CoV-2, a través del método RT-LAMP comparado con el método estándar RT-qPCR. Resultados: el método LAMP detectó ~18 copias del gen N, en 30 min, demostrando un límite de detección similar al obtenido con el método estándar, en un menor tiempo y una concordancia en RT-LAMP del 100 % con los resultados. Conclusiones: RT-LAMP es un método sensible, específico y rápido que puede ser empleado para el diagnóstico de la enfermedad COVID-19.

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: 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. Methods: 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. Results: 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. Conclusions: RT-LAMP is a sensitive, specific, and rapid method that can be used as a diagnostic aid of COVID-19 disease.

RESUMO Introdução: o coronavírus SARS-CoV-2, causador da doença de COVID-19, tornou-se um problema de saúde pública global que requer a implementação de testes diagnósticos rápidos e sensíveis. Objetivo: avaliar e comparar o limite de detecção do método LAMP em relação ao método padrão e aplicar o método RT-LAMP para o diagnóstico de SARS-CoV-2 em amostras clínicas de pacientes colombianos. Métodos: foi realizado um estudo descritivo e transversal, analisando um total de 25 amostras de hissopado nasofaríngeo, incluindo amostras negativas e positivas para SARS-CoV-2, pelo método RT-LAMP em comparação ao método RT-qPCR padrão. Resultados: o método LAMP detectou ~18 cópias do gene N, em 30 min., demonstrando um limite de detecção semelhante ao obtido com o método padrão, em um tempo menor e uma concordância no RT-LAMP de 100% com os resultados. Conclusões: RT-LAMP é um método sensível, específico e rápido que pode ser usado para o diagnóstico da doença de COVID-19.

Comparision on the effect of RT-LAMP and LAMP method for detection of Mycobacterium tuberculosis / 中国人兽共患病学报

We compared the RT-LAMP,LAMP and L-J for detecting MTB to provide the rapid diagnosis method for tu-berculosis.In this assay,NTM and other common respiratory bacteria were used to detect specificity,Mycobacterium tubercu-losis specific products were identified by restriction enzyme digestion.To detect sensitivity,we used RT-LAMP,LAMP and L-J to detect 100 cases sputum specimens from patients with tuberculosis and 22 cases control sputum specimens,the 10 times dilution of 1 ng/μL H37Rv standard strains was used to detect RT-LAMP limit.The results showed that the positive rate of RT-LAMP,LAMP and L-J were 100%,92%,88%.RT-LAMP and L-J,RT-LAMP and LAMP were statistically signifi-cant,RT-LAMP had 10 times higher sensitivity than LAMP,RT-LAMP not only to identify viable but also capable of detec-ting a single copy of MTB.So RT-LAMP is superior to LAMP and L-J and is practical for use in primary medical care institu-tion or peripheral laboratory.

A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification

BACKGROUND: Porcine Deltacoronavirus (PDCoV) is a newly emerged enteropathogenic coronavirus that causes diarrhea and mortality in neonatal piglets. PDCoV has spread to many countries around the world, leading to significant economic losses in the pork industry. Therefore, a rapid and sensitive method for detection of PDCoV in clinical samples is urgently needed. RESULTS: In this study, we developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay specific for nucleocapsid gene to diagnose and monitor PDCoV infections. The detection limit of RT-LAMP assay was 1 × 101 copies of PDCoV, which was approximately 100-fold more sensitive than gel-based one-step reverse transcription polymerase chain reaction (RT-PCR). This assay could specifically amplify PDCoV and had no cross amplification with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine kobuvirus (PKoV), porcine astrovirus (PAstV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), and porcine circovirus type 2 (PCV2). By screening a panel of clinical specimens (N = 192), this method presented a similar sensitivity with nested RT-PCR and was 1-2 log more sensitive than conventional RT-PCR in detection of PDCoV. CONCLUSIONS: The RT-LAMP assay established in this study is a potentially valuable tool, especially in low-resource laboratories and filed settings, for a rapid diagnosis, surveillance, and molecular epidemiology investigation of PDCoV infections. To the best of our knowledge, this is the first work for detection of newly emerged PDCoV with LAMP technology.

Establishment of a visualized detection method of Sendai virus by reverse transcription loop-mediated isothermal amplification / 中国实验动物学报

Objective To establish a simple and sensitive detection method of Sendai virus ( SeV ) by reverse transcription loop-mediated isothermal amplification ( RT-LAMP) technique. Methods According to the published Gen-Bank sequences (DQ219803. 1), six pairs of primers were designed targeting the conserved region of SeV. The amplifica-tion products were detected with a LAMP real-time Turbidimeter. (LA-302). Through optimizing the LAMP primers and re-action conditions, a rapid and specific detection method of SeV was established. Meanwhile, the amplified products were colored by fluorescence detection reagent after completion of the reaction, so that the amplification could be visualized and detected by naked eyes. Then, methodological evaluation of the RT-LAMP was tested. Results The method of RT-LAMP showed a highly efficient amplification for SeV viral target gene which was performed at 63℃ for 60 min with the LAMP re-al-time Turbidimeter (LA-302). The detection limit was 2. 1 TCID50, 100 times higher than that of RT-PCR, and no cross-reaction with other RNA and DNA viruses of mice was observed. The results of SeV LAMP reaction was visualized and the tube could be directly observed by naked eyes with the addition of fluorescence detection reagent. The results were consist-ent with the results detected by real-time tubidimeter. 92 clinical samples were detected byRT- LAMP, RT-PCR and indi- rect ELISA, and the coincidence rate was 100%. Conclusions This established SeV RT-LAMP detection method is fast, specific, highly sensitive,easy to perform under simple conditions, and is suitable for rapid detection of Sendai viirus.

Development and evaluation of reverse-transcription loop-mediated isothermal amplification for rapid detection of human immunodeficiency virus type 1.

Purpose: The objective of this study was to establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of human immunodeficiency virus type 1 (HIV-1). Materials and Methods: The HIV-1 integrase gene region was selected because it was a conserved part of the HIV-1 genome. Six primers specific to eight regions of the HIV-1 integrase gene were designed. A total of 171 samples (18 HIV-1 confirmed positive samples and 153 serum specimens were collected in this study) were tested by RT-LAMP and reverse-transcription polymerase chain reaction (RT-PCR). After amplification in an isothermal water bath for 45 min, samples containing HIV-1 generated the expected ladder-like products while other viruses generated no product. Results: The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with RT-PCR. The assay was significantly more sensitive than normal gel-based RT-PCR. Conclusion: Because it is specific and simple, the RT-LAMP assay can be widely applied in clinical laboratories for rapid detection of HIV-1.

Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay / 中国病毒学·英文版

A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV)strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.

A Novel RT-LAMP Assay for Rapid and Simple Detection of Classical Swine Fever Virus / 中国病毒学·英文版

A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV.PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.