Real-time Loop-mediated Isothermal Amplification Assay Research Articles

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 min, 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 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.

Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens

Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.

A portable all-in-one microfluidic device with real-time colorimetric LAMP for HPV16 and HPV18 DNA point-of-care testing

Screening for high-risk human papillomavirus (HPV) infection is one of the most important preventative measures for cervical cancer. However, fast, convenient, and low-cost HPV detection remains challenging, especially in resource-limited settings. Here, we report a portable all-in-one device (PAD) for point-of-care testing (POCT) for HPV16 and HPV18 DNA in cervical swabs. The PAD was engineered to integrate modules for extraction-free sample lysis, loop-mediated isothermal amplification (LAMP) with lyophilized reagent beads, and real-time colorimetric signal sensing into a single miniaturized device, considerably shortening the sample-to-result time to 15 min. The precision liquid handling in the completely sealed microfluidic chip is achieved by a uniquely designed pressure-balanced automatic liquid flow mechanism, thereby eliminating the need for manual manipulation of liquids and thus the risk of biohazards. The PAD employs an improved real-time colorimetric LAMP (rcLAMP) assay with a limit of detection (LOD) of 1 copy/μL, enabled by enhanced assay chemistry to maximize the reaction kinetics. To validate this device for clinical application, we tested 206 clinical cervical swab samples and obtained a sensitivity of 92.1% and a specificity of 99.0%. This custom PAD enabled by microfluidic and electronic engineering techniques can be configured for the simultaneous detection of HPV16 and HPV18 or other pathogens in point-of-care applications.

Real-Time and Rapid Detection of Phytopythium vexans Using Loop-Mediated Isothermal Amplification Assay.

Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, A. M. Lodhi & Levesque is an important waterborne and soil-inhabiting oomycete pathogen causing root and crown rot of various plants including certain woody ornamentals, fruit, and forest trees. Early and accurate detection of Phytopythium in the nursery production system is critical, as this pathogen is quickly transported to neighboring healthy plants through the irrigation system. Conventional methods for the detection of this pathogen are tedious, frequently inconclusive, and costly. Hence, a specific, sensitive, and rapid molecular diagnostic method is required to overcome the limitations of traditional identification. In the current study, loop-mediated isothermal amplification (LAMP) for DNA amplification was developed for the identification of P. vexans. It was evaluated using real-time and colorimetric assays. Several sets of LAMP primers were designed and screened, but PVLSU2 was found to be specific to P. vexans as it did not amplify other closely related oomycetes, fungi, and bacteria. Moreover, the developed assays were sensitive enough to amplify DNA up to 102 fg per reaction. The real-time LAMP assay was more sensitive than traditional PCR and culture-based methods to detect infected plant samples. In addition, both LAMP assays detected as few as 100 zoospores suspended in 100 ml water. These LAMP assays are anticipated to save time in P. vexans detection by disease diagnostic laboratories and research institutions and enable early preparedness in the event of disease outbreaks.

Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Ralstonia solanacearum Species Complex and Phylotype I in Solanaceous Crops

The Ralstonia solanacearum species complex (RSSC) causes bacterial wilt diseases, which affect a wide range of plant hosts, including Solanaceae, and is of major economic importance. Point-of-care (POC) techniques such as loop-mediated isothermal amplification (LAMP) enable the rapid and sensitive detection of plant pathogens and can be deployed directly in the field. For fast and reliable diagnosis on site, we optimized an RSSC-LAMP assay and developed a real-time LAMP assay targeting the phylotype I. Both LAMPs were highly specific, yielding negative results for a wide collection of nontarget strains and positive results for all target strains, except for two particular mulberry strains for the phylotype I assay. These results were supported by an extensive in silico analysis performed on 6,105 genomes of Burkholderiaceae. The two LAMP assays displayed high sensitivity on pure suspensions, with a detection limit at 103 and 104 CFU/ml for phylotype I and the RSSC, respectively. These thresholds correspond to theoretical quantities of as low as 5 and 50 CFU per reaction. Two simplified extraction methods were successfully used to detect the pathogen from different Solanaceae samples. We demonstrated that LAMP assays were operable on solanaceous crops during field surveys and varietal evaluation trials as rapid POC diagnostics tools, which can improve the disease management and control of the RSSC. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 102 genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2-3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions.

Real-Time loop-mediated isothermal amplification assay for rapid detection of Banana bunchy top virus in North-east India

Aim: The study aims to standardize a Real-Time LAMP assay for effective, highly sensitive, and rapid detection of BBTV in North-east India. Methodology: Forty samples of banana showing BBTV like symptoms were collected from Assam, India and subjected to conventional PCR for confirmation. Six sets of BBTV LAMP primers were designed and the PCR positive samples were subjected to Real-Time LAMP assay for detection of BBTV. Finally, a sensitivity test of BBTV LAMP assay and comparison of BBTV LAMP assay with conventional PCR was done using seven 10-fold dilutions of total genomic DNA of leaf samples with the highest dilution starting from 100 ng µl-1. Results: Initially a total of twenty six out of forty banana samples were tested positive for BBTV with conventional PCR method. The Real-Time LAMP assay for BBTV detection resulted in typical sigmoidal amplification curves with the peak values ranging between 8.00 to 12.15 min and annealing derivatives ranging between 83.3oC to 84.3oC in the tested samples. Sensitivity testing and comparison of BBTV Real-Time LAMP assay with conventional PCR revealed that the BBTV LAMP assay could efficiently detect up to 0.0001ng µl-1 of total DNA against 0.01ng µl-1 in conventional PCR. Interpretation: The findings highlight rapid, sensitive, accurate and effective diagnosis of BBTV using Real-Time LAMP method. This method can be preferred over conventional diagnostic techniques like PCR or ELISA for rapid large scale detection of BBTV in banana plants in North-east India. Key words: Banana, Banana bunchy top virus, Rapid detection, Real-Time LAMP assay

Rapid detection of Arothron species by real-time fluorescence and colorimetric loop-mediated isothermal amplification assays targeting the mitochondrial cytochrome b gene

The pufferfish-poisoning epidemic is caused due to many consumers blindly tasting fish of an unknown species or adulterating food ingredients. Arothron is a pufferfish containing tetrodotoxin and saxitoxin in their bodies. Poisoning incidents of caused by Arothron occur occasionally. The purpose of the current study was to develop a simple identification method for Arothron fish based on loop-mediated isothermal amplification (LAMP) using visual colorimetric and real-time fluorescence measurements. The specificity and sensitivity of the techniques were evaluated, and the results proved that identification method was highly specific, with no cross-reaction occurring in non-Arothron fishes. The sensitivity was as low as 1 ng/L in visual colorimetric and real-time fluorescence LAMP assays. Arothron was used to simulate cooked goods and digested vomit to test further if it met the requirements of rapid detection in commercial use and medical testing. The feasibility results proved that the samples could also be successfully detected after cooking or digestion. Real-time LAMP is rapid and colorimetric LAMP is visible to the naked eye. Due to these characteristics, the LAMP method is expected to become an effective tool for species identification to meet the growing demand for food safety control of fish products.

Colorimetric and Real-Time Loop-Mediated Isothermal Amplification (LAMP) for Detection of Loa loa DNA in Human Blood Samples.

Loiasis, caused by the filarial nematode Loa loa, is endemic in Central and West Africa. Loa loa has been associated with severe adverse reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. Diagnosis of loiasis still depends on microscopy in blood samples, but this is not effective for large-scale surveys. New diagnostics methods for loiasis are urgently needed. Previously, we developed a colorimetric high-sensitive and species-specific LAMP for Loa loa DNA detection. Here, we evaluate it in a set of 100 field-collected clinical samples stored as dried blood spots. In addition, Loa loa-LAMP was also evaluated in real-time testing and compared with microscopy and a specific PCR/nested PCR. A simple saponin/Chelex-based method was used to extract DNA. Colorimetric and real-time LAMP assays detected more samples with microscopy-confirmed Loa loa and Loa loa/Mansonella perstans mixed infections than PCR/nested-PCR. Samples with the highest Loa loa microfilariae counts were amplified faster in real-time LAMP assays. Our Loa loa-LAMP could be a promising molecular tool for the easy, rapid and accurate screening of patients for loiasis in endemic areas with low-resource settings. The real-time testing (feasible in a handheld device) could be very useful to rule out high-microfilariae loads in infected patients.

Elimination of Carryover Contamination in Real-Time Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid Detection of the SARS-CoV-2 Virus in Point-of-Care Testing.

Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies/µl (8 copies/reaction) within 45 min of amplification and 2.63 ± 0.17 pg (equivalent to 2.296 × 109 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens.

Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus.

A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material.

Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for Stromatinia cepivora in Response to an Outbreak in Northern Idaho

Stromatinia cepivora is the causal agent of white rot disease of Allium species. In 2018, white rot was observed in Boundary County in Northern Idaho in garlic and onion plants in a variety of home and market garden operations. Because the university diagnostic laboratory for Idaho is situated in Parma within a regulated area for S. cepivora, a point-of-care (POC) assay using real-time loop-mediated isothermal amplification (LAMP) was developed to minimize the amount of material potentially sent to the diagnostic laboratory. The LAMP assay was used with a BioRanger platform, and although the limit of detection meant it was 100 times less sensitive than TaqMan, it was capable of detecting a single sclerotium. This study demonstrates the rapid development and deployment of a POC-suitable LAMP assay. Despite limitations in sensitivity and dynamic range compared with real-time PCR, POC LAMP assays are advantageous where biosecurity concerns prohibit the movement of material suitable for diagnosis as well as facilitating engagement with growers.

A Real-Time Loop-Mediated Isothermal Amplification for Detection of the Wheat Dwarf Virus in Wheat and the Insect Vector Psammotettix alienus.

Wheat dwarf virus (WDV; genus Mastrevirus, family Geminiviridae) is an economically important and widespread pathogen of cereal crops. It causes huge yield loss in wheat because of the unavailability of resistant varieties and rapid transmission by the vector leafhopper, Psammotettix alienus (Dahlb). To monitor and forecast this viral disease, an early diagnosis method is required for WDV detection in both infected plants and the virus vectors. In this study, we developed a real-time loop-mediated isothermal amplification (LAMP) assay for WDV detection. The positive sample could be detected within 28 to 32 min by following a simple, cost-effective procedure. The real-time LAMP assay showed a sensitivity of 2.7 × 105-6 copies/μl for detection and a high specificity for WDV amplification, with a similar accuracy to quantitative PCR. Furthermore, a closed-tube dye method facilitates the inspection of the LAMP reaction and avoids cross-contamination in the detection of the virus. This valuable detection assay could serve as an important tool for diagnosis and forecasting wheat dwarf disease intensity in the field.

Laboratory and Field Evaluations of a Commercially Available Real-Time Loop-Mediated Isothermal Amplification Assay for the Detection of West Nile Virus in Mosquito Pools.

Although the specific cDNA amplification mechanisms of reverse-transcriptase polymerase chain reaction (RT-PCR) and RT loop-mediated isothermal amplification (RT-LAMP) are very different, both molecular assays serve as options to detect arboviral RNA in mosquito pools. Like RT-PCR, RT-LAMP uses a reverse transcription step to synthesize complementary DNA (cDNA) from an RNA template and then uses target-specific primers to amplify cDNA to detectable levels in a single-tube reaction. Using laboratory-generated West Nile virus (WNV) samples and field-collected mosquito pools, we evaluated the sensitivity and specificity of a commercially available WNV real-time RT-LAMP assay (Pro-AmpRT™ WNV; Pro-Lab Diagnostics, Inc., Round Rock, Texas) and compared the results to a validated real-time RT-PCR assay. Laboratory generated virus stock samples containing ≥ 2.3 log10 plaque-forming units (PFU)/ml and intrathoracically inoculated mosquitoes containing ≥ 2.4 log10 PFU/ml produced positive results in the Pro-AmpRT WNV assay. Of field-collected pools that were WNV positive by real-time RT-PCR, 74.5% (70 of 94) were also positive by the Pro-AmpRT WNV assay, resulting in an overall Cohen's kappa agreement of 79.4% between the 2 tests. The Pro-AmpRT WNV assay shows promise as a suitable virus screening tool for vector surveillance programs provided agencies are aware of its characteristics and limitations.

Assessment of a Multiplex LAMP Assay (Eazyplex® CSF Direct M) for Rapid Molecular Diagnosis of Bacterial Meningitis: Accuracy and Pitfalls.

Background: Automated molecular panels are attractive tools for improving early meningitis diagnosis. This study assessed the Eazyplex® CSF direct M panel (EP), a multiplex real-time Loop-Mediated Isothermal Amplification assay. Methods: From December 2016 to December 2019, cerebrospinal fluid (CSF) samples were routinely tested with the EP V1.0. CSF parameters and microbiological and clinical data were retrospectively collected. Results: Out of 230 CSF samples, the EP yielded positive, negative, and invalid results for 32 (13.9%) (16 N. meningitidis, nine S. pneumoniae, two S. agalactiae, two E. coli, two H. influenzae, one L. monocytogenes), 182 (79.1%), and 16 (7%) samples, respectively. Among the positive samples, 14 (44%) remained negative in culture (antibiotic therapy before lumbar puncture (n = 11), meningococcal meningitis (n = 3)). High CSF protein concentrations and cellularity were associated with LAMP inhibition, counteracted by centrifugation. The automated software yielded 13 false positive and five false negative results. Amplification curve analysis was necessary and enabled the attainment of positive (PPA) and negative percentage agreement and positive and negative predictive values of 91.4%, 100%, 100%, and 98.3%. Three false negative results remained (two E. coli and one N. meningitidis). E. coli presented the poorest PPA (50%). Conclusion: This work confirms the strong performance of the EP, of particular interest in cases of antibiotic therapy before lumbar puncture.

One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei

This study highlights the development of a multiplex real-time loop-mediated isothermal amplification assay. The developed assay employed a dual-function oligonucleotide (DFO) which simultaneously monitors the emitted amplification signals and accelerates the amplification process. The DFO was a modification of loop primer (LP); the 5′-end and 3′-end of the LP was tagged with fluorophore and quencher, respectively. The DFO was quenched in its unbound state and fluoresces only when it anneals to the specific target during the amplification process. With the same working mechanism as LP, DFO allowed the detection of target genes in less than 1 h in a real time monitoring system. We demonstrated this detection platform with Burkholderia pseudomallei, the causative agent of melioidosis. An internal amplification control (IAC) was incorporated in the assay to rule out false negative result and to demonstrate that the assay was successfully developed in a multiplex system. The assay was 100% specific when it was evaluated against 96 B. pseudomallei clinical isolates and 48 other bacteria species. The detection limit (sensitivity) of the developed assay was 1 fg/μl of B. pseudomallei genomic DNA and 18.2 CFU/ml at the bacterial cell level. In spiked blood samples, the assay's detection limit was 14 CFU/ml. The assay's diagnostic evaluation showed 100% diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value. An integrated multiplex LAMP and real-time monitoring system was successfully developed, simplifying the workflow for the rapid and specific nucleic acid diagnostic test.

Multiple authentications of high-value milk by centrifugal microfluidic chip-based real-time fluorescent LAMP

Adulteration of food ingredients, particularly replacement of high-value milk with low-cost milk, affects food safety. For rapid and accurate identification of the possible adulterating milk species in an unknown sample, a centrifugal microfluidic chip-based real-time fluorescent multiplex loop-mediated isothermal amplification (LAMP) assay was developed to simultaneously detect milk from cow, camel, horse, goat, and yak. Using precoated primers in different reaction wells, the centrifugal microfluidic chip markedly simplified the detection process and reduced false-positive results. The entire amplification was completed within 90 min with a genomic detection limit of 0.05 ng/µL in cow, camel, horse, and goat milk and 0.005 ng/µL in yak milk. Using simulated adulterated samples for validation, the detection limit for adulterated milk samples was 2.5%, satisfying authentication requirements, as the proportion of adulterated milk higher than 10% affects economic interests. Therefore, this simple, centrifugal, microfluidic chip-based multiplex real-time fluorescent LAMP assay can simultaneously detect common milk species in commercial products to enable accurate labeling.

Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma

ObjectiveThe present study established a real-time loop-mediated isothermal amplification (qLAMP) for rapid detection of human papillomavirus subtype 16 (HPV-16) in oral squamous cell carcinoma (OSCC). MethodsThe qLAMP assay was optimized targeting the HPV-16 E7 gene. The analytical sensitivity and specificity of the assay were determined using HPV-18 (ATCC® 45152D™), HPV-35 (ATCC® 40330™), HPV-43 (ATCC® 40338™) and HPV-56 (ATCC® 40549™) viral strains and oral bacteria. HPV-16 standard curve was constructed for determination of HPV-16 viral load. The diagnostic performance of the assay was evaluated from 63 OSCC patients comprising 63 tissue, 13 saliva and 49 blood samples, in comparison with p16 immunohistochemistry (IHC), in-house PCR and nested PCR assays. ResultsThe detection limit of developed LAMP and PCR assays was 4.68 × 101 and 4.68 × 103 copies/μl, respectively. qLAMP assay enabled detection of positive results as early as 23 min at 67 °C. This assay can detect HPV-16 positivity in 23 % (3/13) saliva and 4.8 % (3/63) tissue samples with the viral load ranging from 4.68 × 101 to 4.68 × 104 copies/μl. HPV-16 positivity was not detected in all the blood samples. The sensitivity and specificity of qLAMP were 100 % in comparison with that of p16 IHC and nested PCR. ConclusionThis study reports for the first time on the use of qLAMP assay for detection of HPV-16 in OSCC in both tissue and saliva as the sample matrix which holds promise in improving the diagnostic application owing to its rapidity, simplicity, high sensitivity and specificity.

Development of a LAMP assay using a portable device for the real-time detection of cotton leaf curl disease in field conditions.

Cotton production is seriously affected by the prevalent cotton leaf curl disease (CLCuD) that originated from Nigeria (Africa) to various parts of Asia including Pakistan, India, China and Philippines. Due to CLCuD, Pakistan suffers heavy losses approximately 2 billion USD per annum. Numerous reports showed that CLCuD is associated with multiple species of begomoviruses, alphasatellites and a single species of betasatellite, that is ‘Cotton leaf curl Multan betasatellite’ (CLCuMuB). The most prevalent form of CLCuD is the combination of ‘Cotton leaf curl Kokhran virus’-Burewala strain (CLCuKoV-Bur) and CLCuMuB. Thus, the availability of an in-field assay for the timely detection of CLCuD is important for the control and management of the disease. In this study, a robust method using the loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CLCuD. Multiple sets of six primers were designed based on the conserved regions of CLCuKoV-Bur and CLCuMuB-βC1 genes. The results showed that the primer set targeting the CLCuMuB-βC1 gene performed best when the LAMP assay was performed at 58°C using 100 ng of total plant tissue DNA as a template in a 25 µl reaction volume. The limit of detection for the assay was as low as 22 copies of total purified DNA template per reaction. This assay was further adapted to perform as a colorimetric and real-time LAMP assay which proved to be advantageously applied for the rapid and early point-of-care detection of CLCuD in the field. Application of the assay could help to prevent the huge economic losses caused by the disease and contribute to the socio-economic development of underdeveloped countries.