Rapid Loop-mediated Isothermal Amplification Research Articles

Development and Validation of the MAST ISOPLEX® VTEC Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) with Inhibition Control (IC) in a Rapid Loop-Mediated Isothermal Amplification (LAMP) Multiplex Assay

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX® VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore–oligonucleotide probe, MAST ISOPLEX® Probes, integrated into the primer set of each target. MAST ISOPLEX® Probes used in the MAST ISOPLEX® VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX® VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.

Development and validation of a rapid loop-mediated isothermal amplification assay for the detection of Chrysomyxa and characterization of Chrysomyxa woroninii overwintering on Picea in China

Chrysomyxa rusts cause significant damage to spruce in both natural forests and plantations. Particularly, Three Chrysomyxa species, Chrysomyxa deformans, Chrysomyxa qilianensis, and Chrysomyxa rhododendri, listed as National Forest Dangerous Pests in China, have severely affected many economically and ecologically important spruce native species in China. Also, Chrysomyxa arctostaphyli, an important plant quarantine fungus, causes a damaging broom rust disease on spruce. Therefore, rapid, and efficient detection tools are urgently needed for proper rust disease detection and management. In this study, a sensitive, genus-specific loop-mediated isothermal amplification (LAMP) assay targeting the ITS-28S rRNA region was developed to detect the presence of Chrysomyxa in spruce needle and bud samples. After optimization and validation, the LAMP assay was found to be sensitive to detect as low as 5.2 fg/µL DNA, making it suitable for rapid on-site testing for rust infection. The assay was also specific to Chrysomyxa species, with no positive signals from other rust genus/species. The application of LAMP in the early detection of rust infections in spruce needles and buds was investigated, and spatial colonization profiles as well as the means of overwintering of Chrysomyxa woroninii in infected buds and branches were verified using the LAMP assay. This LAMP detection method will facilitate further studies on the characteristics of the life cycle and inoculation of other systemic rusts.

A rapid LAMP assay for the diagnosis of oak wilt with the naked eye

BackgroundOak wilt disease, caused by Bretziella fagacearum is a significant threat to oak (Quercus spp.) tree health in the United States and Eastern Canada. The disease may cause dramatic damage to natural and urban ecosystems without management. Early and accurate diagnosis followed by timely treatment increases the level of disease control success.ResultsA rapid assay based on loop mediated isothermal amplification (LAMP) was first developed with fluorescence detection of B. fagacearum after 30-minute reaction time. Six different primers were designed to specifically bind and amplify the pathogen’s DNA. To simplify the use of this assay in the field, gold nanoparticles (AuNPs) were designed to bind to the DNA amplicon obtained from the LAMP reaction. Upon inducing precipitation, the AuNP-amplicons settle as a red pellet visible to the naked eye, indicative of pathogen presence. Both infected and healthy red oak samples were tested using this visualization method. The assay was found to have high diagnostic sensitivity and specificity for the B. fagacearum isolate studied. Moreover, the developed assay was able to detect the pathogen in crude DNA extracts of diseased oak wood samples, which further reduced the time required to process samples.ConclusionsIn summary, the LAMP assay coupled with oligonucleotide-conjugated gold nanoparticle visualization is a promising method for accurate and rapid molecular-based diagnosis of B. fagacearum in field settings. The new method can be adapted to other forest and plant diseases by simply designing new primers.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 nanograms of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

A Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Didymella segeticola Causing Tea Leaf Spot.

Tea leaf spot caused by Didymella segeticola is an important disease that threatens the healthy growth of tea plants (Camellia sinensis) and results in reductions in the productivity and quality of tea leaves. Early diagnosis of the disease is particularly important for managing the infection. Loop-mediated isothermal amplification (LAMP) assay is an efficient diagnostic technique with the advantages of simplicity, specificity, and sensitivity. In this study, we developed a rapid, visual, and high-sensitivity LAMP assay for D. segeticola detection based on sequence-characterized amplified regions. Two pairs of amplification primers (external primers F3 and B3 and internal primers FIP and BIP) were designed based on a specific sequence in D. segeticola (NCBI accession number: OR987684). Compared to common pathogens of other genera in tea plants and other species in the Didymella genus (Didymella coffeae-arabicae, Didymella pomorum, and Didymella sinensis), the LAMP method is specific for detecting the species D. segeticola. The assay was able to detect D. segeticola at a minimal concentration of 1 fg/μL genomic DNA at an optimal reaction temperature of 65 °C for 60 min. When healthy leaves were inoculated with D. segeticola in the laboratory, the LAMP method successfully detected D. segeticola in diseased tea leaves at 72 h post inoculation. The LAMP assays were negative when the DNA samples were extracted from healthy leaves. Leaf tissues with necrotic lesions from 18 germplasms of tea plants tested positive for the pathogen by the LAMP assay. In summary, this study established a specific, sensitive, and simple LAMP method to detect D. segeticola, which provides reliable technical support for estimating disease prevalence and facilitates sustainable management of tea leaf spot.

Evaluation of self-collected nasal, urine, and saliva samples for molecular detection of SARS-CoV-2 using an EUA approved RT-PCR assay and a laboratory developed LAMP SARS-CoV-2 test.

As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.

Development of Molecular-Based Screening Test for Hepatitis B Virus in Human Plasma Samples.

Despite regular administration of hepatitis B virus (HBV) vaccine in several countries, the mortality rate associated with HBV remains significant. The antiviral medications available for the treatment of HBV infection do not suffice for the serious complications related to chronic HBV infection. Additionally, the serological tests fail to detect early viral replication preventing early treatment response. Recently, many studies have demonstrated the significant advantages of loop-mediated isothermal amplification (LAMP) over serological testing and polymerase chain reaction (PCR), for the rapid detection of microbial pathogens. Here we developed a rapid, sensitive, and portable system-integrative LAMP assay for the detection of HBV DNA in plasma samples. The final optimized assay was achieved with an amplification time of less than 45 min at 62°C. The assay showed 100% specificity, 92.20% sensitivity, and a detection limit of 10 copies/µL in 77 HBV-positive plasma samples with known Cq values. Our results showed that the colorimetric LAMP assay is sensitive, efficient, and supremely reliable for rapid detection of HBV, and may be potentially used as a screening test in areas with poor laboratory facilities and limited resource availability.

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.

Isothermal Amplification and Hypersensitive Fluorescence Dual-Enhancement Nucleic Acid Lateral Flow Assay for Rapid Detection of Acinetobacter baumannii and Its Drug Resistance.

Acinetobacter baumannii (A. baumannii) is among the main pathogens that cause nosocomial infections. The ability to rapidly and accurately detect A. baumannii and its drug resistance is essential for blocking secondary infections and guiding treatments. In this study, we reported a nucleic acid fluorescent lateral flow assay (NFLFA) to identify A. baumannii and carbapenem-resistant A. baumannii (CRAB) in a rapid and quantitative manner by integrating loop-mediated isothermal amplification (LAMP) and silica-based multilayered quantum dot nanobead tag (Si@MQB). First, a rapid LAMP system was established and optimised to support the effective amplification of two bacterial genes in 35 min. Then, the antibody-modified Si@MQB was introduced to capture the two kinds of amplified DNA sequences and simultaneously detect them on two test lines of a LFA strip, which greatly improved the detection sensitivity and stability of the commonly used AuNP-based nucleic acid LFA. With these strategies, the established LAMP-NFLFA achieved detection limits of 199 CFU/mL and 287 CFU/mL for the RecA (house-keeping gene) and blaOXA-23 (drug resistance gene) genes, respectively, within 43 min. Furthermore, the assay exhibited good repeatability and specificity for detecting target pathogens in real complex specimens and environments; thus, the proposed assay undoubtedly provides a promising and low-cost tool for the on-site monitoring of nosocomial infections.

Rapid Visual LAMP Method for Detection of Genetically Modified Organisms.

We developed a novel loop-mediated isothermal amplification (LAMP) method using DNA captured on polyacrylamide microparticles (PAMMPs) as templates (PAMMPs@DNA-LAMP) for rapid qualitative detection of genetically modified organisms (GMOs). Here, DNA was extracted by a fast and cost-effective method using PAMMPs. Four LAMP primers were designed for the PAMMPs@DNA-LAMP method to detect the cauliflower mosaic virus 35S (CaMV35S) promotor in GMOs. We thus developed this method for rapid extraction of DNA (5-10 min) and fast amplification of DNA within ∼30 min at a constant temperature of 63 °C. Moreover, the DNA captured by PAMMPs (PAMMPs@DNA) could be effectively detected by both conventional and quantitative PCR (qPCR) and LAMP. The PAMMPs@DNA-LAMP method was validated with high specificity, sensitivity, and performance for practical sample analysis. This assay detected 0.01% target sequences, which had a high specificity like qPCR and better than the conventional PCR (cPCR). Furthermore, PAMMPs@DNA-LAMP was successfully used to extract and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel PAMMPs@DNA-LAMP assay has been developed, which has higher sensitivity and spends less time than the cPCR detection using the conventional DNA extracted process. This method offers a novel approach for rapid detection of GMOs in the field.

Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR.

The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/μL (0.315-1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/μL (2.084-8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778-1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites.

Genetic Authentication of the Medicinal Plant Portulaca oleracea Using a Quick, Precise, and Sensitive Isothermal DNA Amplification Assay

Portulaca oleracea (PO) is a commonly known medicinal crop that is an important ingredient for traditional Chinese medicine (TCM) due to its use as a vegetable in the diet. PO has been recorded to be frequently adulterated by other related species in the market of herbal plants, distorting the PO plant identity. Thus, identification of the botanical origin of PO is a crucial step before pharmaceutical or functional food application. In this research, a quick assay named “loop-mediated isothermal amplification (LAMP)” was built for the specific and sensitive authentication of PO DNA. On the basis of the divergences in the internal transcribed spacer 2 (ITS2) sequence between PO and its adulterant species, the LAMP primers were designed and verified their specificity, sensitivity, and application for the PO DNA authentication. The detection limit of the LAMP assay for PO DNA identification specifically was 100 fg under isothermal conditions at 63 °C for 30 min. In addition, different heat-processed PO samples can be applied for use in PO authentication in the LAMP assay. These samples of PO were more susceptible to the effect of steaming in authentication by PCR than boiling and drying treatment. Furthermore, commercial PO samples pursued from herbal markets were used to display their applicability of the developed LAMP analysis for PO postharvest authentication, and the investigation found that approximately 68.4% of PO specimens in the marketplace of herbal remedies were adulterated. In summary, the specific, sensitive, and rapid LAMP assay for PO authentication was first successfully developed herein, and its practical application for the inspection of adulteration in PO samples from the herbal market was shown. This LAMP assay created in this study will be useful to authenticate the botanical origin of PO and its commercial products.

Optimizing a rapid LAMP assay for discrimination of Drosophila suzukii (Diptera: Drosophilidae) from common drosophilids captured in monitoring traps from the Midwest, United States.

Spotted-wing Drosophila, Drosophila suzukii, is an economically important pest of small fruits worldwide. Currently, the timing of management strategies relies on detection of adult flies captured in baited monitoring traps; however, identifying D. suzukii in trap catch based on morphology can be challenging for growers. DNA-based diagnostic methods such as loop-mediated isothermal amplification (LAMP) have the potential to improve D. suzukii detection. This study evaluated a LAMP assay as a diagnostic tool to discriminate between D. suzukii and closely related drosophilid species found commonly in monitoring traps in the Midwestern United States. Targeting the mitochondrial cytochrome oxidase I (COI) gene, we found the LAMP assay accurately detected D. suzukii with as little as 0.1 ng/μl of DNA at 63 °C for 50 min. Under these optimal incubation conditions, D. suzukii could be discriminated from D. affinis and D. simulans consistently, when specimens collected from liquid monitoring traps were tested independently. Compared to other DNA-based diagnostic tools for D. suzukii, LAMP offers unique benefits: DNA extraction is not required, testing occurs at one temperature in less than 1 h, and positive results are visible as a colorimetric change from pink to yellow. The LAMP assay for D. suzukii can reduce reliance on morphological identification, enhance the adoption of monitoring tools, and improve accuracy of detection. Further optimization can be conducted to evaluate the accuracy and sensitivity of results when a mixture of DNA from both D. suzukii and congener flies are tested in a single LAMP reaction.

Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

Abstract 2115: Zero-waste molecular diagnostics from biopsy needle wash water

Abstract Background: Over the past decade, the WHO classification of brain tumors has increasingly integrated molecular diagnostics into official classification criteria. However, biopsy tissue is precious due to high morbidity associated with repeat biopsies, and usually earmarked for histology, leaving little or no tissue for clinical molecular diagnostics. We have identified that the fluid used to wash biopsy needles after cores contains excess tumor DNA and cells that can be diagnostically useful. Additional research of tumor DNA and tumor tissue recovered from biopsy needle washes provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients, while utilizing material that is otherwise thrown away. Methods: In two cases, surgeons were instructed to save 5-15ml of fluid used to wash biopsy needles. Isothermal targeted amplification of H3.3 and H3.1 histone genes was performed on raw wash fluid. Resulting amplicons were sequenced using an Oxford Nanopore MinION. DNA extracted from wash fluid was subjected to NGS sequencing via the Illumina TruSight Oncology 500 panel and compared to a CLIA certified NGS sequencing results from clinically indicated biopsy tissue. Results: For case 1, 5ul of raw wash fluid was subjected to rapid Loop-mediated Isothermal Amplification (LAMP) targeting the H3.1 and H3.3 K27M histone mutations and immediately sequenced using a MinION sequencer via rapid library preparation. Sequencing revealed a H3.3 K27M histone mutation present in wash fluid at 12% variant allele fraction (VAF). The variant call (99.99% CI) was available 1hr5mins after acquisition of material. For case 2, 5ml of wash fluid was processed using a cell-free DNA extraction kit resulting in ~250ng of recovered DNA. A LAMP assay targeting the H3.3 K27M mutation was performed on extracted DNA from wash fluid. Sequencing revealed an H3.3 K27M histone mutation present at 55% VAF, which was later corroborated using digital droplet PCR. Clinically indicated tissue from both biopsies was submitted for cancer panel sequencing in a CLIA certified setting. DNA extracted from wash fluid from both cases was subjected to Illumina TruSight Oncology 500 panel sequencing. In both cases, the panel was able to corroborate all SNV mutations reported by clinical sequencing that were also covered by the panel. Accrual of needle wash specimens is ongoing, with multiple monthly procedures. Conclusions: Large amounts of diagnostically useful material is present in biopsy needle wash fluid, which is usually discarded. Isothermal assays coupled with MinION sequencing can rapidly identify clinically relevant point mutations directly from wash fluid (e.g. IDH1), and enough DNA was recovered from wash fluid from both case-studies to submit for targeted cancer panel sequencing. These results suggest biopsy needle wash fluid should be considered a first-class diagnostic resource in any cancer. Citation Format: Jack Wadden, Vishal John, Seongbae Kong, Kait Verbal, Amy K. Bruzek, Wajd Al-Holou, Jason A. Heth, Hugh Garton, Carl Koschmann. Zero-waste molecular diagnostics from biopsy needle wash water [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2115.

Authentication of olive oil in commercial products using specific, sensitive, and rapid loop-mediated isothermal amplification.

The online version contains supplementary material available at 10.1007/s13197-023-05726-y.

Specificity and Sensitivity of a Rapid LAMP Assay for Early Detection of Emerald Ash Borer (Agrilus planipennis) in Europe

Buprestids are an emerging threat to broadleaf forests across the world. Species such as emerald ash borer (EAB, Agrilus planipennis) seriously threaten ash (Fraxinus spp.) in North America and Europe. As it continues spreading west from European Russia, native European ash populations will suffer dramatic losses. Due to their cryptic lifestyle of the egg and larval stages on developing bark and vascular tissue, buprestids and other wood borers can be difficult to detect. Early detection tools are vital to implement fast eradication measures, and prevent the establishment of invasive species populations. Detection methods using polymerase chain reaction (PCR) assays to target specific taxa can be extremely timely to obtain results especially since samples need to be transported to the laboratory first. However, loop-mediated isothermal amplification (LAMP) eDNA assays are highly specific and sensitive providing results within 30 min after sample extraction. In this study, we investigated the specificity and sensitivity of an EAB LAMP assay as an early detection tool in Europe. The assay was specific to EAB when tested against 12 European Agrilus spp., five buprestids, two Scolytinae, and five cerambycids (n = 24). The LAMP assay sensitivity amplified DNA from a concentration as low as 0.02 pg/µL. These results demonstrate that the LAMP assay is a highly specific, sensitive tool that can be used to detect and monitor EAB in European forests and urban settings.

Development of a simple, rapid, and sensitive molecular diagnostic assay for cholera

Cholera continues to inflict high rates of morbidity and mortality. Prompt identification of cholera cases facilitates rapid outbreak responses in the short term while providing reliable surveillance data to guide long-term policies and interventions. Microbiological stool culture, the current recognized gold standard for diagnosing cholera, has significant limitations. Rapid diagnostic tests (RDTs) represent promising alternatives for diagnosing cholera in areas with limited laboratory infrastructure. However, studies conducted with the current cholera RDTs demonstrated wide variations in sensitivity and specificity. To address this gap in the diagnosis of cholera, we developed a simple, rapid, and sensitive diagnostic assay, "Rapid LAMP based Diagnostic Test (RLDT)." With a novel, simple sample preparation method directly from the fecal samples along with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, cholera toxin gene (ctxA) and O1 (O1rfb) gene could be detected in less than an hour. Cholera RLDT assay is cold chain and electricity-free. To avoid any end-user bias, a battery-operated, handheld reader was used to read the RLDT results. The performance specifications of the cholera RLDT assay, including analytical sensitivity and specificity, were evaluated using direct fecal samples, dried fecal samples on filter paper, and environmental water samples spiked with cholera strain. The limit of detection (LOD) was ~104 CFU/gm of stool for both ctxA and O1 genes, corresponding to about 1 CFU of Vibrio cholerae per reaction within 40 minutes. The LOD was 10 bacteria per ml of environmental water when tested with RLDT directly, without enrichment. Being simple, RLDT has the potential to be applied in resource-poor endemic settings for rapid, sensitive, and reliable diagnosis of cholera.

Accelerated loop-mediated isothermal amplification method for the rapid detection of Streptococcus suis serotypes 2 and 14 based on single nucleotide polymorphisms.

Streptococcus suis serotypes 2 and 14 are the most prevalent zoonotic strains. The establishment of a sensitive and extremely accurate method for point-of-care testing for Streptococcus suis serotype 2 and 14 strains is highly desirable. In this study, a loop primer probe-introduced loop-mediated isothermal amplification assay was developed to differentiate Streptococcus suis serotypes 2 and 14 based on SNP (single nucleotide polymorphism). The specific fluorescent probes were designed for the SNP site specific for serotype 2 and 14 Streptococcus suis cpsK genes, and the loop primer probe-introduced loop-mediated isothermal amplification (LAMP) assay was developed using the specific cleavage properties of the RNase H2 enzyme. Rapid and efficient LAMP assays were realized through the use of loop forward primers and stem forward primers. The results showed that the amplification reaction can be performed efficiently at 59°C. The results can be real-time detected or judged using a smartphone and a 3D-printed visualization cassette. The sensitivity of the LAMP assay can reach 18.4 CFU within 40 minutes. The detection rate of the assay system was evaluated using 19 clinical samples with suspected Streptococcus suis infection, and the detection rate was consistent with the sequencing method, suggesting that the test is highly practical. The LAMP assay for Streptococcus suis serotypes 2 and 14 established in this study has strong specificity, high sensitivity, and simple operation, while the reaction can be performed at an isothermal temperature and is not dependent on complex instruments or professional operators, making it suitable for field testing.

Noninvasive Approach of Plasmodium falciparum Molecular Detection for Malaria Surveillance in Malaria Endemic Areas in Cameroon

Accurate, cost-effective, and noninvasive alternative molecular methods are needed for detecting low malaria parasitemia. The currently-used nested polymerase chain reaction (nPCR) requires blood as well as skilled personnel in order to minimise the risk of bloodborne disease transmission. Therefore, this study is aimed at assessing the accuracy of a noninvasive and more affordable malaria diagnosis with saliva using the loop-mediated isothermal amplification (LAMP) technique. A cross-sectional study was conducted in the Centre and Southwest regions of Cameroon. Matched blood and saliva samples collected from symptomatic and asymptomatic participants were tested for malaria using rapid diagnostic tests, microscopy, PCR, and LAMP. Statistics were performed using R studio software at 95% confidence interval. A total of 100 participants (65% symptomatic and 35% asymptomatic) aged between 1 and 74 years with a balanced gender distribution ratio of 1.08 were included in our study. The prevalence of malaria was 61%, 57%, 59%, 42%, 35%, 17%, and 16% for blood-RDT, blood-PCR, blood-LAMP, blood-RT-LAMP, saliva-PCR, saliva-RT-LAMP, and saliva-LAMP, respectively. Both saliva and blood showed a sensitivity of 43.90% and respective specificities of 68.75% and 57.62%. When using RT-LAMP, sensitivities of 49.38% and 48.21% and specificities of 94.11% and 66.67% were recorded for saliva and blood, respectively. Sensitivities of 70.23% and 73.49% and specificities of 62.5% and 76.47% were recorded, respectively, for saliva-LAMP and saliva-RT-LAMP when compared to saliva-PCR as the gold standard. Saliva-LAMP and saliva-RT-LAMP had a fair agreement (к = 0.221 and 0.352, respectively) with saliva-PCR. Homemade LAMP and RT-LAMP technologies match the WHO recommendations and after proper validation in a larger sample size, could serve for malaria diagnosis in developing countries.