Isothermal Amplification Method Research Articles

A colorimetric reverse transcription-loop mediated isothermal amplification (RT-LAMP) method for the rapid detection of SARS-CoV-2 in Thailand

A colorimetric reverse transcription-loop mediated isothermal amplification (RT-LAMP) method for the rapid detection of SARS-CoV-2 in Thailand

Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Detection of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in Inactivated Fermented Milk Using Fluorescence Quantitative Loop-Mediated Isothermal Amplification

Currently, no effective method exists to detect and monitor fermentation probiotics and evaluate the quality of inactivated fermented milk. Therefore, in this study, a fluorescence quantitative loop-mediated isothermal amplification (FQ-LAMP) method was developed to detect Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The specificity of LAMP primers for L. bulgaricus and S. thermophilus was verified using S-type amplification curves and a single peak at approximately 88.568°C and 83.704°C of the melting curves, respectively. The lowest quantification limits of FQ-LAMP for the two strains in inactivated fermented milk were 8.1 × 103 CFU/g (170 fg/μL) and 6.8 × 103 CFU/g (170 fg/μL), respectively. FQ-LAMP was used to analyse 40 inactivated fermented milk samples from six randomly selected brands. The logarithmic concentration of S. thermophilus in all products was between 7.482 and 8.936. The logarithmic concentration of L. bulgaricus ranged from 4.590 to 8.277, with no detectable L. bulgaricus in three samples. FQ-LAMP has the potential as a rapid, specific, and accurate method for detecting and monitoring L. bulgaricus and S. thermophilus in inactivated fermented milk during their shelf life.

A simple graphene oxide-based DNA purification strategy for plant pathogen detection.

The on-site molecular detection of plant pathogens is particularly important for the development of sustainable agriculture. Extracting DNA from plant tissues, microbes or coexisting environments is complex, labor-intensive and time-consuming. To facilitate this process, we propose a DNA purification strategy based on graphene oxide (GO). The excellent adsorption ability of GO was verified by visualizing changes in its microscopic surface and macroscopic mixture. To further optimize the DNA purification, we determined the optimal GO concentration and treatment time at 95 °C (2 mg mL-1 and 2 min, respectively). We confirmed that our strategy is effective on plant tissues and various microorganisms, and that the obtained DNA can be directly used for polymerase chain reaction amplification. Combining the proposed GO-based DNA purification method with the loop-mediated isothermal amplification method is superior, in terms of the required steps, time, cost and detection effect, to the cetyltrimethylammonium bromide method and a commercial kit for detecting plant pathogens. We present a feasible, rapid, simple and low-cost DNA purification method with high practical value for scientific applications in plant pathogen detection. This strategy can also provide important technical support for future research on plant-microbial microenvironments. © 2024 Society of Chemical Industry.

Detection of Parasites in the Field: The Ever-Innovating CRISPR/Cas12a.

The rapid and accurate identification of parasites is crucial for prompt therapeutic intervention in parasitosis and effective epidemiological surveillance. For accurate and effective clinical diagnosis, it is imperative to develop a nucleic-acid-based diagnostic tool that combines the sensitivity and specificity of nucleic acid amplification tests (NAATs) with the speed, cost-effectiveness, and convenience of isothermal amplification methods. A new nucleic acid detection method, utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, holds promise in point-of-care testing (POCT). CRISPR/Cas12a is presently employed for the detection of Plasmodium falciparum, Toxoplasma gondii, Schistosoma haematobium, and other parasites in blood, urine, or feces. Compared to traditional assays, the CRISPR assay has demonstrated notable advantages, including comparable sensitivity and specificity, simple observation of reaction results, easy and stable transportation conditions, and low equipment dependence. However, a common issue arises as both amplification and cis-cleavage compete in one-pot assays, leading to an extended reaction time. The use of suboptimal crRNA, light-activated crRNA, and spatial separation can potentially weaken or entirely eliminate the competition between amplification and cis-cleavage. This could lead to enhanced sensitivity and reduced reaction times in one-pot assays. Nevertheless, higher costs and complex pre-test genome extraction have hindered the popularization of CRISPR/Cas12a in POCT.

A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.

Fabrication of a Microfluidic Test Device with a 3D Printer and Its Combination with the Loop Mediated Isothermal Amplification Method to Detect Streptococcus pyogenes.

New rapid, reliable, and cost-effective alternative systems are needed for the rapid diagnosis of Streptococcus pyogenes. The aim of this study was to fabricate a microfluidic test device to detect Streptococcus pyogenes by combining the Loop-mediated isothermal amplification method via a 3D printer. Microfluidic test devices were designed in CATIA V5 Release 16 software, and data were directly transferred to a 3D printer and produced using the FDM method with biocompatible PLA filament. The S. pyogenes ATCC 19615 and different ATCC strains was used. Following identification by classical culture methods, a 0.5 McFarland suspension was prepared from the colonies, and DNA isolation was performed from this liquid by a boiling method. S. pyogenes specific speB gene was used to desing LAMP primer sets in PrimerExplorer V5 software and tested on a microfluidic device. LAMP reactions were performed on microfluidic device and on a microcentrifuge tube separately. Both results were analyzed using the culture method as the standard method to diagnostic values. Melting curve analysis of the amplicons of the LAMP reactions performed on a LightCycler 480 system to detect amplification. Among the 50 positive and 100 negative samples, only four samples were found to be false negative by LAMP reaction in a microcentrifuge tube, while eight samples were found to be false negative by LAMP reaction on a microfluidic device. Six samples were found to be false positive by the LAMP reaction in the microcentrifuge tube, while ten samples were found to be false positive by the LAMP reaction on a microfluidic chip. The sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP reactions performed in the microcentrifuge tube and on the microfluidic device were 92-84%, 94-90%, 88.46-80.77%, and 95.92-91.84%, respectively. The limit of detection (LOD) was found to be the same as 1.5 × 102 CFU/mL and the limit of quantification (LOQ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 2.46 × 102-7.4 × 102 CFU/mL, respectively. Cohen's kappa (κ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 0.620-0.705, respectively. In conclusion, our data showed that the LAMP method can be combined with microfluidic test device to detect S. pyogenes, this microfluidic device can be manufactured using 3D printers and results are close to gold standard methods. These devices can be combined with LAMP reactions to detect different pathogens where resources are limited and rapid results are required.

Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

The Present and Future Landscapes of Molecular Diagnostics.

Nucleic acid testing is the cornerstone of modern molecular diagnostics. This review describes the current status and future directions of molecular diagnostics, focusing on four major techniques: polymerase chain reaction (PCR), next-generation sequencing (NGS), isothermal amplification methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods. We explore the advantages and limitations of each technique, describe how each overlaps with or complements other techniques, and examine current clinical offerings. This review provides a broad perspective into the landscape of molecular diagnostics and highlights potential future directions in this rapidly evolving field.

Current Trends in RNA Virus Detection via Nucleic Acid Isothermal Amplification-Based Platforms.

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

Dissecting Diagnostic and Management Strategies for Plant Viral Diseases: What Next?

Recent advancements in molecular biology have revolutionized plant disease diagnosis and management. This review focuses on disease diagnosis through serological techniques, isothermal amplification methods, CRISPR-based approaches, and management strategies using RNA-based methods. Exploring high-throughput sequencing and RNA interference (RNAi) technologies like host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), this review delves into their potential. Despite the precision offered by RNAi in pest and pathogen management, challenges such as off-target effects and efficient dsRNA delivery persist. This review discusses the significance of these strategies in preventing aphid-mediated plant virus transmission, emphasizing the crucial role of meticulous dsRNA design for effective viral RNA targeting while minimizing harm to plant RNA. Despite acknowledged challenges, including off-target effects and delivery issues, this review underscores the transformative potential of RNA-based strategies in agriculture. Envisaging reduced pesticide dependency and enhanced productivity, these strategies stand as key players in the future of sustainable agriculture.

The Identification of Yak Meat Using Loop-Mediated Isothermal Amplification Method Coupled with Hydroxy Naphthol Blue for the Prevention of Food Fraud

Objective. Yak is found in the Qinghai-Tibet Plateau and represents a meat of high nutritional value and good flavor. However, the production of yak is limited, and yak meat adulteration is a growing concern in the marketplace. To protect consumer rights and prevent unfair competition, it is necessary to use an efficient assay to identify the species of yak meat rapidly and accurately being sold. Methods. Loop-mediated isothermal amplification (LAMP) combined with hydroxy naphthol blue (HNB) was used to identify potential adulterants. The specificity and sensitivity tests of yak-derived components were carried out to achieve the monitoring of yak-derived components. Results. The optimal color development was achieved with an external primer-to-internal primer ratio of 400 nmol/L : 1200 nmol/L, 1.5 mmol/L dNTP, and 0.32 U/μL Bst DNA polymerase with 5 mmol/L MgSO4 at 62°C amplification temperature. The detection sensitivity of LAMP-HNB for yak-derived DNA was up to 1 pg/μL. Conclusion. The LAMP-HNB assays provided a valuable tool for the identification of yak gene from adulterated meat. This further enabled the LAMP-HNB assay to be applicable in the identification of other meat products.

A Real-time Quantitative Loop Mediated Isothermal Amplification Assay Targeting com1 Gene of Coxiella burnetii

Background & Objectives: The present study designed a loop mediated isothermal amplification (LAMP) method to rapidly detect Coxiella burnetii (C. burnetii) and develop a sensitive Real-time quantitative LAMP (Q-LAMP) assay to quantify Q fever. Materials & Methods: Primers were specifically designed for use in targeting the com1 conserved gene of C. burnetii to carry out LAMP detection of the agent causing Q fever. After obtaining the LAMP reaction, the sensitivity of the method was assessed by preparing a serial ten-fold dilution of a plasmid carrying com1 gene. Results: The assay’s sensitivity for the visual detection of changes in turbidity or turbidimeter and electrophoresis of agarose gel were 0.4 fg and 0.04 fg, respectively. Hence, the lower limit of detection was 120 and 12 copies of the gene detected in 60 min. The results of this study were indicative of the simplicity, rapidness, sensitivity, and specificity of the LAMP assay for C. burnetii detection and a probable improvement of the diagnostic procedure used in clinical laboratories. Conclusion: The assay specificity was assessed using Coxiella genomic DNA and a panel of Gram-negative and Gram-positive bacteria. The LAMP assay was shown to be highly specific for detection of Coxiella without any observable amplification products from non-Coxiella organisms.

Tetrahedral DNA nanostructures enhance transcription isothermal amplification for multiplex detection of non-coding RNAs

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers.

Rapid Visual Detection of Methicillin-Resistant Staphylococcus aureus in Human Clinical Samples via Closed LAMP Assay Targeting mecA and spa Genes.

The emergence of antimicrobial resistance (AMR), particularly methicillin-resistant Staphylococcus aureus (MRSA), poses a significant global health threat as these bacteria increasingly become resistant to the most available therapeutic options. Thus, developing an efficient approach to rapidly screen MRSA directly from clinical specimens has become vital. In this study, we establish a closed-tube loop-mediated isothermal amplification (LAMP) method incorporating hydroxy-naphthol blue (HNB) colorimetric dye assay to directly detect MRSA from clinical samples based on the presence of mecA and spa genes. In total, 125 preidentified S. aureus isolates and 93 clinical samples containing S. aureus were sourced from the microbiology laboratory at Hamad General Hospital (HGH). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were computed based on conventional PCR. The assay demonstrated 100% specificity, 91.23% sensitivity, 0.90 Cohen Kappa (CK), 100% PPV, and 87.8% NPV for the clinical samples, while clinical isolates exhibited 100% specificity, 97% sensitivity, 0.926 CK, 100% PPV, and 88.89% NPV. Compared to cefoxitin disk diffusion, LAMP provided 100% specificity and sensitivity, 1.00 CK, and 100% for PPV and NPV. The study revealed that the closed-tube LAMP incorporating (HNB) dye is a rapid technique with a turnaround time of less than 1 h and high specificity and sensitivity.

Rapid Visual Detection of Elite Erect Panicle Dense and Erect Panicle 1 Allele for Marker-Assisted Improvement in Rice (Oryza sativa L.) Using the Loop-Mediated Isothermal Amplification Method.

Molecular-assisted breeding is an effective way to improve targeted agronomic traits. dep1 (dense and erect panicle 1) is a pleiotropic gene that regulates yield, quality, disease resistance, and stress tolerance, traits that are of great value in rice (Oryza sativa L.) breeding. In this study, a colorimetric LAMP (loop-mediated isothermal amplification) assay was developed for the detection of the dep1 allele and tested for the screening and selection of the heavy-panicle hybrid rice elite restorer line SHUHUI498, modified with the allele. InDel (Insertion and Deletion) primers (DEP1_F and DEP1_R) and LAMP primers (F3, B3, FIP, and BIP) for genotyping were designed using the Primer3 Plus (version 3.3.0) and PrimerExplore (version 5) software. Our results showed that both InDel and LAMP markers could be used for accurate genotyping. After incubation at a constant temperature of 65 °C for 60 min with hydroxynaphthol blue (HNB) as a color indicator, the color of the LAMP assay containing the dep1 allele changed to sky blue. The SHUHUI498 rice line that was detected in our LAMP assay displayed phenotypes consistent with the dep1 allele such as having a more compact plant architecture, straight stems and leaves, and a significant increase in the number of effective panicles and spikelets, demonstrating the effectiveness of our method in screening for the dep1 allele in rice breeding.

Monkeypox Diagnosis in Clinical Settings: A Comprehensive Review of Best Laboratory Practices.

An outbreak of monkeypox (Mpox) was reported in more than 40 countries in early 2022. Accurate diagnosis of Mpox can be challenging, but history, clinical findings, and laboratory diagnosis can establish the diagnosis. The pre-analytic phase of testing includes collecting, storing, and transporting specimens. It is advised to swab the lesion site with virus transport medium (VTM) containing Dacron or polyester flock swabs from two different sites. Blood, urine, and semen samples may also be used. Timely sampling is necessary to obtain a sufficient amount of virus or antibodies. The analytical phase of infectious disease control involves diagnostic tools to determine the presence of the virus. While polymerase chain reaction (PCR) is the gold standard for detecting Mpox, genome sequencing is for identifying new or modified viruses. As a complement to these methods, isothermal amplification methods have been designed. ELISA assays are also available for the determination of antibodies. Electron microscopy is another effective diagnostic method for tissue identification of the virus. Wastewater fingerprinting provides some of the most effective diagnostic methods for virus identification at the community level. The advantages and disadvantages of these methods are further discussed. Post-analytic phase requires proper interpretation of test results and the preparation of accurate patient reports that include relevant medical history, clinical guidelines, and recommendations for follow-up testing or treatment.

Rapid Detection of Colletotrichum siamense from Infected Tea Plants Using Filter-Disc DNA Extraction and Loop-Mediated Isothermal Amplification.

The pathogen Colletotrichum siamense causes tea anthracnose, resulting in economic losses to the Chinese tea industry. To effectively diagnose this pathogen in the field, we developed a loop-mediated isothermal amplification (LAMP) method using highly specific primers with a sensitivity of 1 pg/μl designed for amplifying the CAL gene, which was 10 times higher than that of conventional PCR. Additionally, to improve the method for obtaining DNA samples required for on-site diagnosis, we used the filter-disc DNA extraction method, which does not require special instruments and can be completed in a few minutes, and found that it effectively meets the requirements for the LAMP reaction. Finally, we combined LAMP with a filter-disc DNA extraction method (FDE-LAMP) to diagnose different degrees of disease in inoculated samples and 20 samples from the field. The results showed that the procedure had sufficient sensitivity for pathogen detection. Therefore, the FDE-LAMP procedure could greatly contribute to managing and preventing tea anthracnose in the field.

A multiplex recombinase polymerase amplification assay combined with CRISPR/Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus.

Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens that cause acute respiratory tract infections in children. We aimed to develop a rapid method for detecting both pathogens simultaneously. The recombinase polymerase isothermal amplification (RPA) method was combined with the CRISPR/Cas detection system. The assay's specificity and sensitivity were explored by designing RPA primers and CRISPR RNAs (crRNAs) through multi-sequence comparisons, optimizing the reaction conditions, and using a fluorescent reading device. The consistency of the test results of 160 clinical pharyngeal swab samples was studied using quantitative polymerase chain reaction (qPCR) results as a comparative control. RSV and ADV could be detected at levels as low as 104 copies/mL and 103 copies/mL, respectively, within 50 minutes with no cross-reactivity with other similar pathogens. For the clinical samples, compared with the qPCR method, the sensitivities for RSV and ADV were 98.1% and 91.4%, respectively, and the detection specificities were both 100%. The Kappa values were greater than 0.95, suggesting a high degree of consistency. This method for detecting RSV and ADV is rapid, sensitive, and specific. It can accurately detect mixed infections in a timely manner, making it suitable for use in areas with scarce healthcare resources.

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.