Implementation of a Rapid RT-LAMP Saliva-based SARS-CoV-2 Testing Program in the Workplace

Rising SARS-CoV-2 cases, testing delays and the risk of pre-symptomatic and asymptomatic transmission provided the impetus for an in-house rapid testing pro-gram. Employees and their household contacts were encouraged to self-collect saliva samples which were pooled for routine testing using an established colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. In brief, individual or a maximum of four saliva samples were pooled, heat-inactivated to render microorganisms, especially SARS-CoV-2, non-infectious prior to being added to RT-LAMP assay tubes containing either human sample control gene, RNase P or a region of the SARS-CoV-2 gene, ORF1ab. During the second wave of SARS-CoV-2 infections in November 2020, two samples from an employee and a member of their household tested positive via RT-LAMP within two days of each other. A delayed clinical qRT-PCR test confirmation of both individuals 5 days later underscores the power of routine rapid testing with within-the-hour turnaround times. Workplace rapid testing programs using RT-LAMP are flexible in their design, have a reduced cost compared to qRT-PCR, may involve non-invasive self-saliva collection for increased safety for the testing personnel, and can be performed with minimal training.

Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pseudomonas fluorescens in Raw Milk

Raw milk is susceptible to microbial contamination during transportation and storage. Pseudomonas fluorescens producing heat-resistant enzymes have become the most common and harmful psychrophilic microorganisms in the cold chain logistics of raw milk. To rapidly detect P. fluorescens in raw milk, the protease gene aprX was selected as a detection target to construct a set of primers with strong specificity, and a loop-mediated isothermal amplification (LAMP) assay was established. The detection thresholds of the LAMP assay for pure cultured P. fluorescens and pasteurized milk were 2.57 × 102 and 3 × 102 CFU/mL, respectively. It had the advantages over conventional method of low detection threshold, strong specificity, rapid detection, and simple operation. This LAMP assay can be used for online monitoring and on-site detection of P. fluorescens in raw milk to guarantee the quality and safety of dairy products.

Rapid diagnosis of Toxoplasma gondii using loop-mediated isothermal amplification assay in camels and small ruminants

Background This study was conducted to detect the presence of T. gondii in milk and blood samples using three different assays: enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification assay (LAMP). Whole blood, serum, and milk samples were collected from goats (n = 156), sheep (n = 261), and camels (n = 108) in different governorates in Egypt from December 2019 to February 2021 and screened by ELISA for anti-Toxoplasma IgG antibodies before DNA extraction. The target T. gondii DNA gene was detected and evaluated using the LAMP assay compared to PCR. Results T. gondii antibodies were found in milk and serum samples at the rates of (29.26%) and (36.58%) in camels, (34.18%) and (35.89%) in sheep, and (33.7%) and (36.36%) in goats, respectively. Similar to PCR, the percentages of LAMP tests for the detection of the T. gondii DNA gene in milk and blood samples of camels, sheep, and goats were (4.8, 14.63), (6.83, 7.69), and (7.79, 9.09), respectively. LAMP's sensitivity for detecting T. gondii in milk and blood samples, which was identical to that of PCR, was 100%. Conclusions The findings clearly demonstrated that there were no variations in T. gondii detection capabilities in milk and blood samples from various animals using both PCR and LAMP tests. It provides a quick, precise, and sensitive method of detecting T. gondii in a variety of samples that may be used both in the field and in laboratory diagnosis.

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

Background Current diagnosis of SARS-CoV-2 infection relies on RNA purification prior to amplification. Typical extraction methods limit the processing speed and turnaround time for SARS-CoV-2 diagnostic testing. Methods Here, we applied reverse transcription loop-mediated isothermal amplification directly onto human clinical swabs samples to amplify the RNA from SARS-CoV-2 swab samples after processing with chelating resin. Results By testing our method on 64 samples, we managed to develop an RT-LAMP assay with 95.9% sensitivity (95% CI 86 to 99.5%) and 100% specificity (95% CI 78.2–100%). Conclusion The entire process including sample processing can be completed in approximately 50 min. This method has promising potential to be applied as a fast, simple and inexpensive diagnostic tool for the detection of SARS-CoV-2.

Loop-mediated isothermal amplification for diagnosing marine pathogens in tissues of Crassostrea spp. and white shrimp, Litopenaeus vannamei, farmed in Mexico

Loop-mediated isothermal amplification (LAMP) is an accurate, sensitive, rapid, and easy-to-perform method for gene amplification under isothermal conditions, and it has served as a powerful diagnostic tool. In this study, we used LAMP to develop a diagnostic protocol for detecting Vibrio parahaemolyticus and white spot syndrome virus in whiteleg shrimp, and Perkinsus spp. in Crassostrea spp. in Mexico. These pathogens are associated with different diseases and are considered a threat in the aquaculture industry. Infected and uninfected oysters and shrimp were obtained from farms in the northwest coast of Mexico to standardize the LAMP assay. We determined the candidate target genes in the first-round analysis of many sets of primers, and then we chose a set of primers that successfully amplified with Mexican samples. We optimized the LAMP reactions for each pathogen with the chosen primer sets using temperature gradients from 61 to 65 ºC, DNA concentrations from 2.5 pg to 250.0 ng, and reaction times from 10 to 60 min. This study established a diagnostic procedure for detecting pathogens in oysters and shrimp from Mexico. Early diagnosis and treatment of pathogens can immensely reduce disease transmission in aquaculture farms.

Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

A loop-mediated isothermal DNA amplification (LAMP) assay for detection of the clubroot pathogen Plasmodiophora brassicae

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or polymerase chain reaction (PCR)-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, being accurate and convenient to visualize. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots and seeds. This method can detect P. brassicae at a minimal amount of 1 fg plasmid DNA or 10 resting spores in the soil. Compared to conventional PCR, the LAMP was more sensitive in detection P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate and easy-to-use LAMP assay to detect P. brassicae, which will facilitate to plan sustainable clubroot management.