Loop-mediated Isothermal Amplification Assay Research Articles

A novel fluoro colorimetric Loop mediated isothermal amplification (LAMP) assay for detection of Trichomonasvaginalis

BackgroundTrichomoniasis is a curable, non-viral, sexually transmitted infection. Early diagnosis and treatment of cases can prevent complications and further spread of infection. Rapid diagnostics tests, which can be performed on-site, will help in early diagnosis. The study aims to develop a rapid diagnostic test based on the principle of fluoro-colorimetric LAMP for detecting Trichomonas vaginalis (TV). Materials and methodsT. vaginalis was grown in a modified CPLM medium, and DNA was extracted. Three pairs of LAMP primers targeting the actin gene were designed using the primerexplorer V.5 online tool. The LAMP assay was standardized for temperature and time. To determine the LAMP assay's detection limit, diluted TV DNA and spiked urine samples were used. Conventional PCR was done using previously published primers and compared with LAMP results. The sensitivity and specificity to detect TV from clinical specimens were assessed. ResultsThe optimum performance of the LAMP assay was determined to be 63 °C for 60 min and terminated at 80 °C for 5 min. The LAMP assay could detect 60 fg/μl of diluted TV DNA and up to 1 parasite/ml of spiked samples. The assay was 1000 times more sensitive than PCR. The LAMP assay was 100% sensitive and specific with crude extract, and reactions were visually discernible. Interpretation & conclusionsThe LAMP assay developed in the study is easy to perform and interpret, affordable, rapid, and highly sensitive to detect T. vaginalis. It is ideally suited for the point-of-care test, as it fulfills WHO's recommended ASSURED characteristics.

A Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Bremia lactucae in the field.

A real-time loop-mediated isothermal amplification (LAMP) assay for the detection of Bremia lactucae, the causal pathogen of Lettuce Downy Mildew (LDM), was developed and validated to aid in-field detection of airborne inoculum. Assay specificity was confirmed against a range of other pathogenic oomycete and fungal spp and sensitivity of the assay for the detection of DNA extracted from sporangia was evaluated. The B. lactucae LAMP assay reliably detected DNA equivalent to 1 spore/reaction (16.7 pg DNA/reaction). Following extraction of DNA from rotorod air-samplers to which sporangial suspensions were added, the assay reliably detected 25 sporangia/rotorod. Detection of airborne inoculum of B. lactucae collected through the season from air-samplers deployed in field plots infected with B. lactucae and also in commercial lettuce fields in Scotland over two growing seasons was assessed. The method can be deployed on samples collected from commercial lettuce production to inform disease management strategies and limit the use of unecessary prophylactic pesticide applications.

Loop-Mediated Isothermal Amplification and Lateral Flow Immunochromatography Technology for Rapid Diagnosis of Influenza A/B.

Influenza viruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Rapid detection of influenza viruses is essential for accurate diagnosis and the initiation of appropriate treatment. We developed a loop-mediated isothermal amplification and lateral flow assay (LAMP-LFA) capable of simultaneously detecting influenza A and influenza B. Primer sets for influenza A and influenza B were designed to target conserved regions of segment 7 and the nucleoprotein gene, respectively. Optimized through various primer set ratios, the assay operated at 62 °C for 30 min. For a total of 243 (85 influenza A positive, 58 influenza B positive and 100 negative) nasopharyngeal swab samples, the performance of the influenza A/B multiplex LAMP-LFA was compared with that of the commercial AllplexTM Respiratory Panel 1 assay (Seegene, Seoul, Korea). The influenza A/B multiplex LAMP-LFA demonstrated a specificity of 98% for the non-infected clinical samples, along with sensitivities of 94.1% for the influenza A clinical samples and 96.6% for the influenza B clinical samples, respectively. The influenza A/B multiplex LAMP-LFA showed high sensitivity and specificity, indicating that it is reliable for use in a low-resource environment.

A Foldable Thermoplastic Microdevice Integrating Isothermal Amplification and Schiff-Reaction-Based Colorimetric Assay for the Detection of Infectious Pathogens

In this study, we introduce a plastic-based foldable microdevice that integrates loop-mediated isothermal amplification (LAMP) and a colorimetric assay based on the Schiff reaction to detect the genes of infectious bacteria. The device comprises two sides: a sample zone containing amplification chambers and a detection zone for the colorimetric assay. The detection zone contains poly(methyl methacrylate) structures for transferring the colorimetric reagent-soaked glass micro-fiber paper into the sample chambers. Specific genes of Staphylococcus aureus (S. aureus) and Streptococcus pneumoniae (S. pneumoniae), the most common bacterial infection causes, were amplified by LAMP assay. The S. aureus gene was detected up to 10 fg/μL and the S. pneumoniae gene up to 0.1 pg/μL. The amplified target genes were visually identified using a colorimetric assay with Schiff’s reagent, which showed clear color discrimination through a reaction with aldehyde groups derived from the DNA in the amplicons. The introduced method, integrating amplification and detection processes in a single device, is expected to be utilized in point-of-care testing analysis for the simple and rapid detection of infectious pathogens.

Development of a real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primers for rapid detection of rubella virus

Rubella virus infection during early pregnancy sometimes causes severe birth defects termed congenital rubella syndrome. Although there are safe and effective live-attenuated vaccines, rubella has only been certified as eliminated in the Americas within the six World Health Organization regions. Rubella remains an endemic disease in many regions, and outbreaks occur wherever population immunity is insufficient. There are two main methods for diagnosis of rubella: detection of anti-rubella IgM antibodies by enzyme immunoassay and detection of the viral genome by real-time RT-PCR. Both of these methods require substantial time and effort. In the present study, a rapid rubella detection assay using real-time fluorescent reverse transcription loop-mediated isothermal amplification with quenching primers was developed. The time required for the new assay was one-half that required for a real-time RT-PCR assay. The assay had 93.6% positive percent agreement and 100% negative percent agreement for clinical specimens compared with the real-time RT-PCR assay. The new assay is considered useful for diagnosis of rubella in areas where rubella is endemic.

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.

Rapid on-site authentication of three cockle species using real-time loop-mediated isothermal amplification (LAMP) assay

Cockles, crucial for commercial fishing and aquaculture, emerge as highly marketable shellfish. However, the morphological similarities among various cockle species are frequently exploited for shellfish fraud. In this study, we developed LAMP assays (end-point LAMP and real-time LAMP) to detect three commercially important cockle species: Tegillarca granosa, Anadara broughtonii, and Anadara kagoshimensis. The primers were designed based on mitochondrial DNA and used to develop LAMP assays. Exclusivity was assessed using 17 shellfish species, and no cross-reactivity was confirmed. The estimate of the detection limit was determined to be 0.0001 ng. The established assays were further refined to create an on-site real-time LAMP assay by combining fast DNA isolation with a real-time fluorometer for field application. The effectiveness of the on-site real-time LAMP assay was successfully verified using 38 commercial samples, with analysis accuracy confirmed using a DNA extraction kit. Consequently, the on-site real-time LAMP assay can complete the whole procedure, including DNA extraction, real-time LAMP reaction, and data analysis within a 45 min timeframe, maintaining the accuracy of detecting three cockle species from processed cockle samples in the field. Our method proves valuable for detecting shellfish fraud by identifying cockle species using samples of both raw and processed products in the field.

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites

BackgroundNatural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. MethodsBy using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 minutes, with the assays performed at 65°C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. ResultsThere was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. ConclusionThis is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus

BackgroundThe monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. ResultsHere, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SignificanceThe device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

The Utilization of the SaLux19-Based Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid and Sensitive Identification of Minute Amounts of a Biological Specimen

Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.

Development of a reverse transcription loop-mediated isothermal amplification assay with novel quantitative pH biosensor readout method for SARS-CoV-2 detection.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a molecular amplification method that can detect SARS-CoV-2 in a shorter time than the current gold-standard molecular diagnostic reverse transcription-polymerase chain reaction (RT-PCR). However, previously developed RT-LAMP assays have mostly relied on highly subjective visual colorimetric interpretation. In this study, an RT-LAMP assay was developed with quantitative measurement of reaction pH using a novel portable pH biosensor compared to qualitative colorimetric interpretation and gel electrophoresis, with 57 clinical COVID-19 samples used for validation of the test. The LoD of the assay is 103 copies/μL. The highest sensitivity was found in the qualitative methods (93.75%), while the highest specificity and likelihood ratio was found in the pH sensor (87.5% and 6.72). On the sensor measurement, a significant difference (p < 0.0001) was observed between the average pH of the RT-PCR (+) COVID-19 (6.15 ± 0.27), while the average pH of the RT-PCR (-) samples (6.72 ± 0.22). Correlation analysis revealed a strong correlation (r = 0.78, p < 0.0001) between the Ct values obtained from RT-PCR with the biosensor pH readout. RT-LAMP with the quantitative pH sensor readout method has the potential to be further developed as an objective molecular assay for rapid and simple detection of SARS-CoV-2.

Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94fg/µLand 25.8fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12h enrichment. The study highlights the importance of short (6-12h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

MinION Sequencing of Fungi in Sub-Saharan African Air and a Novel LAMP Assay for Rapid Detection of the Tropical Phytopathogenic Genus Lasiodiplodia.

To date, there have been no DNA-based metabarcoding studies into airborne fungi in tropical Sub-Saharan Africa. In this initial study, 10 air samples were collected onto Vaseline-coated acrylic rods mounted on drones flown at heights of 15-50 meters above ground for 10-15 min at three sites in Ghana. Purified DNA was extracted from air samples, the internal transcribed spacer (ITS) region was amplified using fungal-specific primers, and MinION third-generation amplicon sequencing was undertaken with downstream bioinformatics analyses utilizing GAIA cloud-based software (at genus taxonomic level). Principal coordinate analyses based on Bray-Curtis beta diversity dissimilarity values found no clear evidence for the structuring of fungal air communities, nor were there significant differences in alpha diversity, based on geographic location (east vs. central Ghana), underlying vegetation type (cocoa vs. non-cocoa), or height above ground level (15-23 m vs. 25-50 m), and despite the short flight times (10-15 min), ~90 operational taxonomic units (OTUs) were identified in each sample. In Ghanaian air, fungal assemblages were skewed at the phylum taxonomic level towards the ascomycetes (53.7%) as opposed to basidiomycetes (24.6%); at the class level, the Dothideomycetes were predominant (29.8%) followed by the Agaricomycetes (21.8%). The most common fungal genus in Ghanaian air was cosmopolitan and globally ubiquitous Cladosporium (9.9% of reads). Interestingly, many fungal genera containing economically important phytopathogens of tropical crops were also identified in Ghanaian air, including Corynespora, Fusarium, and Lasiodiplodia. Consequently, a novel loop-mediated isothermal amplification (LAMP) assay, based on translation elongation factor-1α sequences, was developed and tested for rapid, sensitive, and specific detection of the fungal phytopathogenic genus Lasiodiplodia. Potential applications for improved tropical disease management are considered.

A Multiplex Fluorescence of Loop Primer Upon Self-Dequenching Loop-Mediated Isothermal Amplification Assay for the Detection of Epstein-Barr Virus and Human Parvovirus B19 in Clinical Transplant Samples.

Viral infections are major causes of mortality in solid-organ and hematopoietic stem cell transplant recipients. Epstein-Barr virus (EBV) and Parvovirus B19 (B19V) are among the common viral infections after transplantation and were recommended for increased screening in relevant guidelines. Therefore, the development of rapid, specific, and cost-effective diagnostic methods for EBV and B19V is of paramount importance. We applied Fluorescence of Loop Primer Upon Self-Dequenching Loop-mediated Isothermal Amplification (FLOS-LAMP) for the first time to develop a novel multiplex assay for the detection of EBV and B19V; the fluorophore attached to the probe are self-quenched in unbound state. After binding to the dumbbell-shaped DNA target, the fluorophore is dequenched, resulting in fluorescence development. The novel multiplex FLOS-LAMP assay was optimized by testing various ratios of primer sets. This novel assay, with great specificity, did not cross-react with the common virus. For the detection of EBV and B19V, the limits of detection could reach 969 and 798 copies/μL, respectively, and the assay could be completed within 25 min. Applying this novel assay to detect 200 clinical transplant individuals indicated that the novel assay had high specificity and good sensitivity. We developed multiplex FLOS-LAMP assay for the detection of EBV and B19V, which has the potential to become an important tool for clinical transplant patient screening.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

LAMP assay to detect Elsinoë necatrix; an important Eucalyptus shoot and leaf pathogen.

Eucalyptus scab and shoot malformation caused by Elsinoë necatrix is an emerging disease and a serious threat to the global commercial forestry industry. The disease was first discovered in North Sumatra, Indonesia and now requires a simple and effective method for early pathogen detection. In this study, a rapid and sensitive Loop-mediated isothermal amplification (LAMP) assay was developed for E. necatrix. A unique region in a secondary metabolite gene cluster was used as target for the assay. To test robustness of the assay, LAMP amplification was verified in 15 strains of E. necatrix. A specificity test against 23 closely related Elsinoë species and three fungal species commonly isolated on Eucalyptus showed that the LAMP assay exclusively identified E. necatrix isolates. The assay had a high level of sensitivity, able to detect 0.01 ng (approximately 400 target copies) of pure E. necatrix DNA. Furthermore, using a simple DNA extraction method, it was possible to use this assay to detect E. necatrix in infected Eucalyptus leaves.

Food Authentication: The Detection of Arbutus unedo and Olea europaea Leaves as an Admixture of Oregano Using LAMP- and Duplex LAMP-Based Test Systems with Lateral-Flow Assays

The Mediterranean herb oregano is one of the most frequently adulterated foods. Often morphologically similar leaf material is used as a filler, which can generally be detected using DNA-based methods. Loop-mediated isothermal amplification (LAMP) has high potential for point-of-care testing as it requires only a simple device for sample incubation and is less sensitive to inhibition by co-isolated metabolites compared to conventional PCRs (polymerase chain reactions). In this work, we have developed two LAMP assays for the specific detection of the adulterants olive (Olea europaea) and strawberry tree (Arbutus unedo). The combination with a rapid isolation protocol and LFAs (Lateral-flow assays) as a visualization technique provides a reliable indication of possible adulteration. It has also been shown that it is possible to estimate the level of contamination and to perform the LAMP/LFA assay with DNA isolation in less than 30 min. As a further option, a duplex LAMP/LFA assay was developed that allows both contaminants to be detected in parallel, making the rapid test system even more cost-effective and user-friendly.

Development of nicotiana-specific molecular markers and their application in a loop-mediated isothermal amplification assay

An effective identification method for detecting illegal goods involving raw tobacco material is crucial for tobacco monopolies to conduct surveillance. We developed Nicotiana-specific molecular markers to determine whether seized goods contain raw tobacco material. The sequence data for genes related to the nicotine metabolism pathway and genomic data from the public Solanaceae database were used to establish Nicotiana-specific molecular markers. These markers were determined by experimentally verifying 17 types of nontobacco plant material and 91 types of tobacco material belonging to 11 sections of 3 subgenera. Two reliable Nicotiana-specific markers, Ntsp027 and Ntsp151, were selected from among the 209 newly developed markers. The results indicated that the primers corresponding to these two markers can amplify the target fragments in the 91 types of Nicotiana material without amplification of any PCR products in the 17 types of non-Nicotiana material. Furthermore, utilizing the marker Ntsp151, we verified the efficacy of the loop-mediated isothermal amplification (LAMP) assay in authenticating tobacco material. The identification of 21 tea-cigarette products via the combination of GC‒MS, a Nicotiana-specific molecular marker and LAMP methods underscores the utility of Nicotiana-specific DNA markers in determining whether illegal goods contain raw tobacco material. Our results indicate an impressive accuracy rate of 100%, which is consistent with the reliability assessment, underscoring the accuracy of these markers in effectively identifying tobacco material. Our findings can significantly augment the capacity for surveillance and anticounterfeiting efforts by aiding the fight against illicit trade and ensuring the integrity of all tobacco-related products in the market.

Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.

Evaluation of Loop-Mediated Isothermal Amplification Assay in Diagnosing Tuberculous Meningitis.

Resource-limited settings like India need a simple, quick, and temperature-independent point-of-care diagnostic test that can diagnose tuberculous meningitis (TBM) at the earliest. A prospective study was carried out at a tertiary care center in North India wherein 50 subjects suspected of TBM were recruited and followed up for six months between January 2019 and December 2020. The aim was to evaluate the performance of loop-mediated isothermal amplification (TB-LAMP) in diagnosing TBM as compared to a composite reference standard (CRS), mycobacteria growth indicator tube 960 (MGIT 960) culture, and GeneXpert®. Out of 50 patients, 32 were TBM cases (64%), and 18 were non-TBM cases (36%). The sensitivity of TB-LAMP and GeneXpert® for TBM diagnosis against CRS was 53% (17/32) for both, and the specificity was 78% (14/18) and 89% (16/18), respectively. On comparing TB-LAMP against GeneXpert® for TBM diagnosis, the two methods had almost perfect agreement (Cohen's kappa=0.83) with statistical significance (p<0.001). The performance of TB-LAMP assay is comparable to GeneXpert® in diagnosing TBM, and it may be used as a substitute for CSF GeneXpert® in resource-limited settings.