Loop-mediated Isothermal Amplification Reaction Research Articles

Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

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.

LAMP based electrochemical sensor for extraction-free detection of Listeria monocytogenes in food samples

Listeria monocytogenes (LMO) represents one of the most serious hazards for food health due to the high mortality rate and its ability to survive at limiting conditions. In this study, an electrochemical sensor for the detection of LMO in food samples using loop mediated isothermal amplification (LAMP) technique is presented. The extraction step for the release of the nucleic acids of the sample was simplified using three different alternatives (QuickExtract buffer lysis, heating method and an extraction-free option). The three protocols were tested with the LAMP reaction in seven different food matrices (Dairy milk, fresh cheese, ham, hummus, lettuce, milk powder and pâté) and were able to detect 1 cfu/25 g as stipulated by the food safety regulations. It was proved that all three alternatives were compatible with the electrochemical detection using methylene blue redox-active molecule in dairy milk samples. The final validation of the sensor was carried out with the extraction-free option in nine different food matrices, which were able to detect 1 cfu/25 g of food sample in all cases. This validation demonstrates the feasibility of the sensor for its application in the food industry as a point-of-test solution.

Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/μL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics.

Establishment of a real‐time fluorescence and visual colorimetric detection method for Staphylococcus aureus based on loop‐mediated isothermal amplification

AbstractA loop‐mediated isothermal amplification (LAMP) method combined with calcein was established for the detection of Staphylococcus aureus. LAMP primers were designed targeting the ASL18_04625 species‐specific gene screened through bioinformatics and PCR analysis. The target gene and detection method demonstrated 100% specificity for S. aureus and no cross‐reactions with other pathogens. For LAMP reactions, the results could be directly observed with the naked eye or evaluated by S‐shaped amplification curves of fluorescence signals. The assay's detection limit was 3.395 × 10−4 ng/μL genomic DNA or 3.21 CFU/mL pure bacterial culture. Furthermore, the target bacteria of 8.5 × 105–8.5 × 100 CFU/mL could be accurately detected in spiked milk samples, and the overall detection process could be completed within 40 min. This method improved the accuracy and convenience for S. aureus detection and provied a dependable tool for the rapid screening of S. aureus.

Computer Program for Primer Design for Loop-Mediated Isothermal Amplification (LAMP)

Introduction. To date, numerous methods of nucleic acid amplification have been proposed, and each method has a number of advantages and disadvantages. One of the most popular methods is Loop-mediated isothermal AMPlification (LAMP). Unlike thermocyclic reactions, such as PCR (polymerase chain reaction), which require three temperature changes and expensive equipment, in LAMP, the entire reaction takes place at one and the same temperature and at the maximum rate possible. An important component of LAMP-amplification is primers (usually 20–25 nucleotides), which need to be matched to a specific part of the nucleotide sequence. It is known that DNA sequence contains four nucleotides: A — adenine and T — thymine, G — guanine and C — cytosine. There is a huge variety of permutations of these nucleotides, and it is practically impossible to analyze such a large amount of data manually. Therefore, there is a need to use modern computer technologies. More than 150 computer programs have been proposed for the design of PCR primers, while for LAMP-primers there are less than 10 of them, and each of them has a number of drawbacks, e.g., in terms of the length of the analyzed site. Therefore, this work is aimed at developing a new domestic computer program for the design of specific primers for LAMP.Materials and Methods. The primer search algorithm was based on a linear search for a substring in a string, taking into account the criteria of primer selection for LAMP. The program complex of LAMP-primer design was implemented in Python programming language. The bioPython library was used to work with various DNA and RNA, and the Qt framework was used to develop the interface.Results. A modification of the direct sampling method using a stencil approach was proposed, taking into account the GC composition and annealing temperature of primers depending on their structure. A software package with a friendly interface was developed. It took into account the design criteria of primers: certificates of registration of computer programs (LAMPrimers iQ No. 2022617417 dated April 20, 2022, LAMPrimers iQ_loop No. 2023662840 dated June 14, 2023) were received. The program is in the public domain at https://github.com/Restily/LAMPrimers-iQDiscussion and Conclusion. The developed software packages can be used for research and analysis in molecular biology and genetics, to create diagnostic test systems that provide high sensitivity and reliability of detection of specific DNA and RNA. The software packages can be used in research institutes and laboratories engaged in the amplification of nucleic acids. The results of evaluating the selected sets of primers for the LAMP reaction were tested, and the effectiveness of working sets using the LAMPrimers iQ program was experimentally proven by the example of the detection of genetic material of the SARS-CoV-2 coronavirus.

[Capacity building in schistosomiasis control institutions in China: a cross-sectional study].

To understand the current status of capacity building in schistosomiasis control institutes in schistosomiasis-endemic provinces (municipality, autonomous region) of China. The responsibilities and construction requirements of various schistosomiasis control institutions were surveyed by expert discussions, and field interviews and visits during the period between May and June, 2023, and the questionnaire for capacity maintenance and consolidation in schistosomiasis control institutions was designed. An online questionnaire survey was conducted in county-, municipal-, and provincial-level institutions that undertook schistosomiasis control and surveillance activities through the Wenjuanxing program. The distribution of schistosomiasis control institutions, the status of institutions, departments and staff undertaking schistosomiasis control activities and the translation of scientific researches on schistosomiasis control in China were analyzed. The laboratories accredited by China National Accreditation Service for Conformity Assessment (CNAS) were considered to be capable for testing associated with schistosomiasis control, and the testing capability of schistosomiasis control institutions was analyzed. A total of 486 valid questionnaires were recovered from 486 schistosomiasis control institutions in 12 endemic provinces (municipality, autonomous region) of China, including 12 provincial-level institutions (2.5%), 77 municipal-level institutions (15.8%) and 397 county-level institutions (81.7%). Of all schistosomiasis control institutions, 376 (77.4%) were centers for disease control and prevention or public health centers, 102 (21.0%) were institutions for schistosomiasis, endemic disease and parasitic disease control, and 8 (1.6%) were hospitals, healthcare centers or others. There were 37 713 active employees in the 486 schistosomiasis control institutions, including 5 675 employees related to schistosomiasis control, and the proportions of employees associated with schistosomiasis control among all active employees were 5.9% (231/3 897), 5.5% (566/10 134), and 20.6% (4 878/23 682) in provincial-, municipal-, and county-level institutions, respectively. There were 3 826 full-time employees working in schistosomiasis control activities, with 30.5% (1 166/3 826), 34.6% (1 324) and 34.9% (1 336/3 826) at ages of 40 years and below, 41 to 50 years and over 50 years, and there were 1 571 (41.0%) full-time schistosomiasis control employees with duration of schistosomiasis control activities for over 25 years, and 1 358 (35.5%) employees with junior professional titles and 1 290 with intermediate professional titles (35.5%), while 712 (18.6%) full-time employees working in schistosomiasis control activities had no professional titles. The three core schistosomiasis control activities included snail control (26.3%, 374/1 420), epidemics surveillance and management (25.4%, 361/1 420) and health education (18.8%, 267/1 420) in schistosomiasis control institutions. The Kato-Katz method, miracidium hatching test with nylon gauzes, and indirect haemagglutination assay (IHA) were the most commonly used techniques for detection of schistosomiasis, and there were less than 50% laboratories that had capabilities or experimental conditions for performing enzyme-linked immunosorbent assay (ELISA), dipstick dye immunoassay (DDIA), dot immunogold filtration assay (DIG-FA), loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. During the period from 2018 to 2022, schistosomiasis control institutions had undertaken a total of 211 research projects for schistosomiasis control, with a total funding of 18.596 million RMB, published 619 articles, participated in formulation of 13 schistosomiasis control-related criteria, and applied for 113 schistosomiasis control-related patents, including 101 that were granted, and commercialized 4 scientific research outcomes. The proportion of independent specialized schistosomiasis control institutions is low in schistosomiasis control institutions in China, which suffers from problems of unsatisfactory laboratory testing capabilities, aging of staff and a high proportion of low-level professional titles. More investment into and intensified schistosomiasis control activities and improved capability building and talent cultivation in schistosomiasis control institutions are recommended to provide a powerful support for high-quality elimination of schistosomiasis in China.

Design and evaluation of loop-mediated isothermal amplification for rapid detection of Enterocytozoon bieneusi

Enterocytozoon bieneusi is one of the most prevalent microsporidia species, responsible for more than 90% of human and animal microsporidiosis. Microsporidia species, particularly E. bieneusi, are frequently reported from waterborne and foodborne outbreaks. Therefore, early detection is crucial in clinics and outbreak investigations. This study aimed to design a loop-mediated isothermal amplification (LAMP) for rapid detection of E. bieneusi. Total DNA was extracted from 30 E. bieneusi –positive samples, which had been confirmed with nested PCR. LAMP primers were designed based on the identical fragment of small subunit ribosomal RNA (SSU rRNA) gene. LAMP reactions were performed at 63 °C for 60 min. The sensitivity and specificity of the assay were analyzed and the results of amplification were compared to real-time PCR. Our results showed that the LAMP assay successfully amplified 25/30 (83.3%) samples. The specificity results indicated no false positive with other microorganisms. Furthermore, the LAMP method exhibited a sensitivity (limit of detection, LoD) as low as 34 ag/μL of total DNA. Compared to the LAMP assay, real-time PCR was able to detect all 30 nested PCR-positive samples. Our findings showed that the LAMP assay was able to detect 83.3% of E. bieneusi-positive samples. Although the current assay was not able to detect all nested PCR-positive samples, the lack of need for specific instruments, rapid processes, and high specificity makes LAMP assay a suitable tool for screening.

A novel loop-mediated isothermal amplification (LAMP) assay to diagnose feline panleukopenia.

Protoparvovirus carnivoran1, known commonly as feline panleukopenia virus (FPV) is a highly contagious and environmentally stable parvovirus of domestic as well as wild felids. A rapid and robust diagnostic tool will aid in implementing prompt treatment and control measures. A loop-mediated isothermal amplification (LAMP) as a point-of-care diagnostic tool for diagnosing feline panleukopenia was standardised using faecal samples of cats. The assay will reduce the cost and time required to diagnose feline panleukopenia. A set of two outer primers (F3 and B3) and two inner primers (FIP and BIP) were designed to target the viral polypeptide (VP2) gene of FPV. Optimisation of the LAMP reaction was done at 60 °C for one hour after an initial denaturation at 95 °C for five minutes. Visualisation of the result based on the addition of SYBR Green 1 dye offered an easy and reliable diagnosis. The detection limit of the standardised LAMP assay was as low as 1.25 ng/μl of the target DNA. Species specificity of the LAMP primers revealed no amplification of the non-target DNA of any other species except that of the canine parvovirus DNA template. DNA extracted from 100 PCR-positive and 20 PCR-negative faecal samples were subjected to the standardised assay and compared with PCR. Analysis of the results revealed that the LAMP assay was 100% sensitive and 90% specific compared to PCR. The LAMP assay could be a reliable tool for the point-of-care diagnosis of feline panleukopenia in limited resource settings.

Rapid detection of a downy mildew pathogen, Peronospora destructor, in infected onion tissues and soils by loop-mediated isothermal amplification.

Downy mildew of onion caused by a soil-inhabiting water mold, Peronospora destructor is one of the most devastating disease that can destroy entire onion fields in a matter of days. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay that allows rapid detection of P. destructor by visual inspection. The internal transcribed spacer 2 (ITS2) region of P. destructor was used to design primer sets for LAMP reactions. Optimal temperature and incubation time were determined for the most efficient primer set. In the optimized condition, the LAMP assay exhibited at least 100 times more sensitivity than conventional PCR, detecting fentogram levels of P. destructor genomic DNA (gDNA). Detection of the pathogen from a small number of spores without gDNA extraction further confirmed the high sensitivity of the assay. For specificity, the LAMP assay was negative to gDNA of other fungal pathogens that cause various diseases on onion and oomycetes, while the assay was positive to gDNA extracted from onion tissues showing the typical downy mildew symptoms. Finally, we examined the efficacy of the LAMP assay in detection of P. destructor in soils. Soils collected from onion fields that had been contaminated with P. destructor were solarized for 60 days. While the LAMP assay was negative to the solarized soils, we were able to detect P. destructor that oversummers in fields. The LAMP assay developed in this study enables rapid detection and diagnosis of downy mildew of onion in infected tissues and in soil.

Versatility of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) from diagnosis of early pathological infection to mutation detection in organisms.

Loop-mediated isothermal amplification (LAMP) is a rapid, state-of-the-art DNA amplification technology, used primarily for the quick diagnosis and early identification of microbial infection, caused by pathogens such as virus, bacteria and malaria. A target DNA can be amplified within 30min using the LAMP reaction, taking place at a steady temperature. The LAMP method uses four or six primers to bind eight regions of a target DNA and has a very high specificity. The devices used for conducting LAMP are usually simple since the LAMP method is an isothermal process. When LAMP is coupled with Reverse Transcription (RT), it allows direct detection of RNA in a sample. This greatly enhances the efficiency of diagnosis of RNA viruses in a sample. Recently, the rampant spread of COVID-19 demanded such a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. Loop-mediated isothermal amplification(LAMP) assays are not only used for the detection of microbial pathogens, but there are various other applications such as detection of genetic mutations in food and various organisms. In this review, various implementations of RT-LAMP techniques would be discussed.

Detecting Fasciola hepatica and F. gigantica microRNAs  using loop-mediated isothermal amplification (LAMP)

Fascioliasis is a parasitic infection typically caused by two common parasites of class Trematodo, genus Fasciola, named Fasciola hepatica and F. gigantica. The widespread appearance of these species in water and food makes fascioliasis become a global zoonotic disease that affects 2.4 million people in more than 75 countries worldwide. Typically, F. hepatica and F. gigantica can be recognized with parasitological techniques to detect Fasciola spp. eggs, immunological techniques to detect worm-specific antibodies, or molecular techniques for instance polymerase chain reactions to detect parasitic genomic DNA. Recently, miRNAs have been recognised a key regulator and potential diagnostic biomarkers of diseases, including parasitic infection. An isothermal PCR called LAMP (loop-mediated isothermal amplification) is rapid, sensitive, and this amplification is very extensive, making it well-suited for field diagnostics. LAMP reaction for miRNA detection has been introduced and is able to detect miRNA in the range between 1.0amol and 1.0pmol, showing high selectivity to differentiate one miRNA sequence from others. Here, we introduced a modified LAMP to detect a typical miRNA of both F. hepatica and F. gigantica. Our method does not demand an initial heating step and the reactions have a high sensitivity even 1,000 times higher in comparison to that reported in previous studies. These results create a promising technique basis for some novel and simple device to diagnose fascioliasis and other parasitic diseases at point-of-care.

Simultaneous detection of multiple spoilage bacteria using a fluorogenic loop-mediated isothermal amplification-based multi-sample microfluidic chip

Spoilage bacteria, which ubiquitously contribute to food spoilage, pose a global public health risk. In this study, we developed a centrifugal microfluidic chip integrating fluorogenic loop-mediated isothermal amplification (LAMP) to detect six important spoilage bacteria: Bacillus cereus, Clostridium botulinum, Photobacterium phosphoreum, Pseudomonas putida, Streptococcus agalactiae, and Shewanella putrefaciens. The LAMP reaction components were pre-mixed (primers pre-embedded in each reaction chamber of the chip) and evenly pipetted into the two inlet holes, enabling the simultaneous analysis of six genetic targets from a single sample. This facilitates the automated detection of up to four samples simultaneously using a single chip. The LAMP reactions were conducted in a 5-μL volume at 65 °C for 60 min, achieving limits of detection ranging from 10−1 to 10−2 pg/μL of bacterial genomic DNA. Almost all coefficients of variation for time-to-positive values consistently remained under 10% when genomic DNA was used at concentrations within the limits of detection for the six bacteria. This demonstrated excellent reproducibility across replicates. The clinical sensitivity and specificity for field sample detection were 93.3% and 97.5%, respectively. This method offers ease of operation, rapid analysis, and minimal reagent usage and has great potential for the on-site detection of multiple spoilage bacteria.

Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing.

Moraxella catarrhalis (M. catarrhalis) was an important pathogen closely associated with respiratory tract infections. We employed the loop-mediated isothermal amplification (LAMP) coupled with nanoparticle-based lateral flow biosensor (LFB) and fluorescence testing technique for formulating two diagnostic methods for M. catarrhalis detection, termed M. catarrhalis-LAMP-LFB assay and M. catarrhalis-LAMP-FRT, respectively. The M. catarrhalis-LAMP-LFB system incorporated the use of biotin-14-dCTP and a forward loop primer (LF) with a hapten at the 5' end. This design in LAMP reaction enabled the production of double-labeled products that could be effectively analyzed using the lateral flow biosensor (LFB). For the M. catarrhalis-LAMP-FRT assay, the LF was modified with a sequence at 5' end, and a fluorophore, as well as a black hole quencher, were strategically labeled at the 5' end and within the middle of the new LF. The restriction endonuclease Nb.BsrDI could accurately recognize and cleave the newly synthesized double-strand terminal sequences, resulting in the separation of the fluorophore from the black hole quencher and releasing fluorescence signals. Both assays have been proven to be highly sensitive and specific, capable of detecting genomic DNA of M. catarrhalis at concentrations as low as 70 fg, with no cross-reactivity observed with non-M. catarrhalis pathogens. Furthermore, both methods successfully identified M. catarrhalis in all clinical samples within 1h that were confirmed positive by real-time PCR, exhibiting superior sensitivity than conventional culture methods. Herein, the newly developed two LAMP-based assays were rapid and reliable for M. catarrhalis detection and hold significant promise for deployment in point-of-care (POC) settings.

Combination-Lock SlipChip Integrating Nucleic Acid Sample Preparation and Isothermal LAMP Amplification for the Detection of SARS-CoV-2.

Nucleic acid analysis with an easy-to-use workflow, high specificity and sensitivity, independence of sophisticated instruments, and accessibility outside of the laboratory is highly desirable for the detection and monitoring of infectious diseases. Integration of laboratory-quality sample preparation on a hand-held system is critical for performance. A SlipChip device inspired by the combination lock can perform magnetic bead-based nucleic acid extraction with several clockwise and counterclockwise rotations. A palm-sized base station was developed to assist sample preparation and provide thermal control of isothermal nucleic acid amplification without plug-in power. The loop-mediated isothermal amplification reaction can be performed with a colorimetric method and directly analyzed by the naked eye or with a mobile phone app. This system achieves good bead recovery during the sample preparation workflow and has minimal residue carryover from the lysis and elution buffers. Its performance is comparable to that of the standard laboratory protocol with real-time qPCR amplification methods. The entire workflow is completed in less than 35 min and the device can achieve 500 copies/mL sensitivity. Thirty clinical nasal swab samples were collected and tested with a sensitivity of 95% and a specificity of 100% for SARS-CoV-2. This combination-lock SlipChip provides a promising fast, easy-to-use nucleic acid test with bead-based sample preparation that produces laboratory-quality results for point-of-care settings, especially in home use applications.

End-point rapid detection of total and pathogenic Vibrio parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) in raw seafood using a colorimetric loop-mediated isothermal amplification-xylenol orange technique.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis in humans worldwide. To ensure seafood safety and to minimize the occurrence of seafood-borne diseases, early detection of total V. parahaemolyticus (pathogenic and non-pathogenic strains) and pathogenic V. parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) is required. This study further improved a loop-mediated isothermal amplification (LAMP) assay using xylenol orange (XO), a pH sensitive dye, to transform conventional LAMP into a one-step colorimetric assay giving visible results to the naked eye. LAMP-XO targeted rpoD for species specificity and tdh, trh1, and trh2 for pathogenic strains. Multiple hybrid inner primers (MHP) of LAMP primers for rpoD detection to complement the main primer set previously reported were designed by our group to maximize sensitivity and speed. Following the standard LAMP protocol, LAMP reaction temperature for rpoD, tdh, trh1, and trh2 detection was first determined using a turbidimeter. The acquired optimal temperature was subjected to optimize six parameters including dNTP mix, betaine, MgSO4, Bst 2.0 WarmStart DNA polymerase, reaction time and XO dye. The last parameter was done using a heat block. The color change of the LAMP-XO result from purple (negative) to yellow (positive) was monitored visually. The detection limits (DLs) of LAMP-XO using a 10-fold serial dilution of gDNA and spiked seafood samples were determined and compared with standard LAMP, PCR, and quantitative PCR (qPCR) assays. Subsequently, the LAMP-XO assay was validated with 102 raw seafood samples and the results were compared with PCR and qPCR assays. Under optimal conditions (65°C for 75 min), rpoD-LAMP-XO and tdh-LAMP-XO showed detection sensitivity at 102 copies of gDNA/reaction, or 10 folds greater than trh1-LAMP-XO and trh2-LAMP-XO. This level of sensitivity was similar to that of standard LAMP, comparable to that of the gold standard qPCR, and 10-100 times higher than that of PCR. In spiked samples, rpoD-LAMP-XO, tdh-LAMP-XO, and trh2-LAMP-XO could detect V. parahaemolyticus at 1 CFU/2.5 g spiked shrimp. Of 102 seafood samples, LAMP-XO was significantly more sensitive than PCR (P < 0.05) for tdh and trh2 detection and not significantly different from qPCR for all genes determined. The reliability of tdh-LAMP-XO and trh2-LAMP-XO to detect pathogenic V. parahaemolyticus was at 94.4% and 100%, respectively. To detect total and pathogenic V. parahaemolyticus, at least rpoD-LAMP-XO and trh2-LAMP-XO should be used, as both showed 100% sensitivity, specificity, and accuracy. With short turnaround time, ease, and reliability, LAMP-XO serves as a better alternative to PCR and qPCR for routine detection of V.parahaemolyticus in seafood. The concept of using a one-step LAMP-XO and MHP-LAMP to enhance efficiency of diagnostic performance of LAMP-based assays can be generally applied for detecting any gene of interest.

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

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

A microfluidic-based quantitative analysis system for the multiplexed genetic diagnosis of human viral infections using colorimetric loop-mediated isothermal amplification.

In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 μL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses.

Optimization of Loop-Mediated Isothermal Amplification Reaction for Detecting Streptococcus pneumoniae Serotypes in Clinical Nasopharynx Swab Samples

Current molecular PCR-based methods for identifying Streptococcus pneumoniae, the primary cause of pneumonia, meningitis, and other invasive diseases, are accurate but require expensive infrastructure and have a long run time, which restricts their use, particularly in developing countries. LAMP, or loop-mediated isothermal amplification, is a low-cost alternative to polymerase chain reaction (PCR) that can be used quickly because the reaction only takes place at a constant temperature. We aimed to develop a LAMP assay for rapid detection of Streptococcus pneumoniae serotypes in nasopharynx swab samples. The LAMP primers were designed using a conserved region of the lytA gene. An incubation time range of 30 to 60 minutes was studied to optimize the LAMP reaction. The real-time fluorescence intensity was monitored during the amplification reaction. Clinical nasopharynx swab samples identified as Streptococcus pneumoniae and their serotypes were tested to evaluate the performance of LAMP. To investigate the specificity of the LAMP, Streptococcus species samples and non-Streptococcus species samples were analyzed. In conclusion, the optimized LAMP assay is capable of detecting Streptococcus pneumoniae and its serotypes in nasopharynx swab samples.

Dry Lamp: A Point of Care Diagnostics for Diagnosis of Bovine Tropical Theileriosis

ABSTRACT Background &amp; objectives: Theileriosis is an important tick-borne hemoprotozoan disease of cattle which causes severe economic loss due to morbidity and mortality. A diagnostic test having high sensitivity, specificity and easy application at the field level is the need of the hour. In this regard Loop mediated isothermal amplification (LAMP) is proven to be a sensitive, easy and time efficient method. One of the major obstacles for the application of LAMP is the difficulty in maintaining the cold chain to preserve reagents. Thus, the challenge is to develop a LAMP kit in a ready-to-use format with dried reagents useful for quick and simple application in field conditions. Methods: The optimized reaction of wet LAMP was followed for the standardization of dry LAMP with certain modifications which are needful. The major modification is vitrification technology of enzyme using trehalose. Results: LAMP assay (dry and wet LAMP) was found to be more sensitive (100%) when compared to microscopy (69.5%) and PCR (86.9%). It was observed that the dry LAMP reaction tubes at room temperature as well as refrigeration temperature provided successful amplification till 7 weeks. Interpretation &amp; conclusion: The drying conditions of LAMP reagents were optimized, and finally managed to dry them in a single reaction tube without reducing the sensitivity. This technology enables us to transport LAMP kits to areas where the cold chain is not easily available.