Enzyme- and label-free cascade isothermal amplification aptasensor for the ultrasensitive detection of ochratoxin A.

BACKGROUND: Ultrasensitive detection is crucial for the early warning and intervention of risk factors, ultimately benefiting the environment and human health. Low levels of ochratoxin A (OTA) present a hidden yet significant threat, and rapid detection via high-performing biosensors is therefore essential. RESULTS: A cascade isothermal amplification aptasensor (CIA-aptasensor) was designed for OTA detection. On the surface of a magnetic bead probe, the OTA level was converted into positively correlated trigger cDNA through its competitive binding with OTA-Apt. The released trigger cDNA activated catalytic hairpin assembly followed by coupling with a hybridization chain reaction to achieve CIA. After adding graphene oxide and SYBR Green I, the background interference was eliminated to specifically obtain OTA-related fluorescence. The ultrasensitive limit of detection was 0.22 pg mL-1, an improvement of 1368-fold over conventional enzyme-linked aptamer sorbent assay by the same OTA-Apt, demonstrating satisfactory reliability and practicability. Thus, the CIA-aptasensor provides an enzyme- and label-free simplified homogeneous system with minimal background interference using isothermal conditions. SIGNIFICANCE: This study provides a polymerase chain reaction-like approach for enhancing the sensitivity and performance of a biosensor, which could be extended for the application of CIA and label-free signaling strategy to other risk factors.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Ultrafast DNA detection based on turn-back loop primer-accelerated LAMP (TLAMP).

Rapid DNA detection is a long-pursuing goal in molecular detection, especially in combating infectious diseases. Loop-mediated isothermal amplification (LAMP) is a robust and prevailing DNA detection method in pathogen detection, which has been drawing broad interest in improving its performance. Herein, we reported a new strategy and developed a new LAMP variant named TLAMP with a superior amplification rate. In this strategy, the turn-back loop primers (TLPs) were devised by ingeniously extending the 5' end of the original loop primer, which conferred the new role of being the inner primer for TLPs while retaining its original function as the loop primer. In theory, based on the bifunctional TLPs, a total of eight basic dumbbell-like structures and four cyclic amplification pathways were produced to significantly enhance the amplification efficiency of TLAMP. With the enhancing effect of TLPs, TLAMP exhibited a significantly reduced amplification-to-result time compared to the conventional six-primer LAMP (typically 1 h), enabling rapid DNA detection within 20 min. Furthermore, TLAMP proved to be about 10 min faster than the fast LAMP variants reported so far, while still presenting comparable sensitivity and higher repeatability. Finally, TLAMP successfully achieved an ultrafast diagnosis of Monkeypox virus (MPXV), capable of detecting as few as 10 copies (0.67copies/µL) of pseudovirus within 20 min using real-time fluorescence assay or within 30 min using a colorimetric assay, suggesting that the proposed TLAMP offers a sensitive, specific, reliable, and, most importantly, ultrafast DNA detection method when facing the challenges posed by infectious diseases.

Rapid Laboratory Diagnosis of Epizootic Hemorrhagic Disease Virus Using a Loop-Mediated Isothermal Amplification Assay.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a molecular diagnostic assay that is particularly useful for the detection of viral diseases of livestock. A major advantage of RT-LAMP is that it can be used either as a rapid field test or as a high-throughput screening tool in veterinary laboratories, with sensitivity comparable to the real-time RT-PCR assay. Unlike conventional or qPCR, RT-LAMP uses a strand displacement polymerase and a set of four to six primers that bind to several regions of the target nucleic acid. Amplification occurs without thermal cycling, and coupled with the numerous primers, RT-LAMP offers a rapid and highly specific molecular assay. In this chapter, we describe the RT-LAMP protocol for the detection of epizootic hemorrhagic disease virus (EHDV) as a low-cost, specific, and sensitive screening tool in veterinary diagnostic laboratories. We also provide guidance on how to adapt the RT-LAMP assay for rapid field testing.

Multiplex fluorescence loop-mediated isothermal amplification with lateral flow assay for rapid simultaneous detection of mecA and nuc genes in methicillin-resistant Staphylococcus aureus.

BACKGROUND: Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), pose a significant threat to public health. Existing detection methods, like cultivation-based techniques, demand significant time and labor, while molecular diagnostic techniques, such as PCR, necessitate sophisticated instrumentation and skilled personnel. Although previous multiplex loop-mediated isothermal amplification assays based on fluorescent dyes (mfLAMP) offer simplicity and cost-effectiveness, they are prone to false-positive results. Therefore, developing a rapid and efficient multiplex assay for high-sensitivity MRSA is imperative to create a practical diagnostic tool for point-of-care testing. RESULTS: Here, we developed a mfLAMP combined with a lateral flow assay (mfLAMP-LFA) for the visual and simultaneous detection of the mecA (PBP2a-specific marker) and nuc (S. aureus-specific marker) genes in MRSA. We optimized mfLAMP-LFA using graphene oxide (GO)-based purification and specific DNA probes and evaluated its sensitivity, specificity, and stability. Utilizing GO to mitigate false-positive results by acting as a trap for free DNA probes, the mfLAMP-LFA method successfully identified mecAf and nucf-probes, exhibiting distinct red, green, and yellow fluorescence signals. The detection sensitivity of the developed mfLAMP-LFA method (1 CFU mL-1 in phosphate-buffered saline (PBS)) was comparable to other highly sensitive MRSA detection methods (1 CFU mL-1 in PBS). Furthermore, the method demonstrated specificity for MRSA, detecting it in irrigation water samples within the desired range and achieving reliable recovery rates from spiked samples. SIGNIFICANCE: This novel strategy is the first to incorporate GO into mfLAMP-LFA, enabling specific and sensitive MRSA detection and advancing rapid bacterial detection. This assay facilitates MRSA diagnostics, contributing to improved public health and food safety by delivering rapid, cost-effective point-of-care results. It enables the simultaneous detection of multiple bacteria, even in irrigation water samples artificially inoculated with MRSA, which contain aerobic bacteria at 2.7 × 102 CFU mL-1.

Multiplex competitive annealing mediated isothermal amplification with high fidelity DNA polymerase (HiFi-CAMP).

Various isothermal amplification methods have been developed for point-of-care testing (POCT) of various infectious diseases. Here, we proposed a novel isothermal amplification method, named as 5'-half complementary primers mediated isothermal amplification (HCPA). Because of the similarity of our method to the previous method competitive annealing mediated isothermal amplification (CAMP) in primer design, we also use the name CAMP for our method. We demonstrated that CAMP is mediated by both a linear isothermal amplification pattern and a loop-mediated isothermal amplification pattern. To improve the specificity and enable multiplex detection, we further developed HiFi-CAMP method that uses a small amount of high-fidelity DNA polymerase to cut HFman probe to release fluorescent signal. The HiFi-CAMP method was demonstrated to have a good specificity and sensitivity, and fast amplification speed in detection of three human respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus A (RSV-A) and influenza A viruses (IAV). When compared with gold standard RT-qPCR assays, the HiFi-CAMP assays showed sensitivities of 90.0 %, 71.4 % and 78.1 %, specificities of 100 %, 100 % and 95.5 %, and consistencies of 93.0 %, 93.3 % and 88.2 % for SARS-CoV-2, RSV-A and IAV, respectively. Furthermore, a duplex HiFi-CAMP assay was also developed to simultaneously detect RSV-A and SARS-CoV-2. The HiFi-CAMP will provide a promising candidate for POCT diagnosis in resource-limited settings.

Molecular detection of toxoplasmosis in wild rats using loop-mediated isothermal amplification assay.

Background and Aim: Toxoplasmosis is caused by the parasite Toxoplasma gondii. Cats are the only known hosts that excrete resistant oocysts. Wild rats serve as crucial reservoirs and intermediate hosts for T. gondii's survival and dissemination. Consuming soil and water containing oocysts can lead to illness. This study aimed to estimate the prevalence of toxoplasmosis in wild rats through molecular detection as an indicator of environmental contamination in Surabaya. Materials and Methods: One hundred rats were collected from the three areas (housing, dense settlements, and traditional markets) and distributed into the five zones: West, East, Central, North, and South of Surabaya. Brain tissue samples were extracted using a Geneaid™ (New Taipei City, Taiwan) DNA isolation kit and analyzed through the loop-mediated isothermal amplification (LAMP) method. Results: The study analyzed brain tissue from 100 wild rats, consisting of 77 Rattus tanezumi and 33 Rattus norvegicus, displaying 30% LAMP positivity. The study revealed that 30% (30/100) of wild rats tested were infected with T. gondii. The molecular prevalence rate in male rats was 32.35% (22/68), compared to females with 25% (8/32). 41.9% of the housing population, 33.3% of traditional markets, and 22.6% of dense settlements had the highest molecular prevalence. The high positive molecular rate at the trapping site can be attributed to cats and dense populations. Conclusion: Thirty percentage wild rats were tested positive for toxoplasmosis in Surabaya, East Java, Indonesia using LAMP method. Implementing strict control and monitoring is crucial in preventing the transmission of diseases from wild rats to humans. It is necessary to carry out further research related to genetic analysis of T. gondii to determine the type of T. gondii that infects animals and humans in Surabaya through bioassay and molecular test.

Loop-Mediated Isothermal Amplification for Detecting Gly-4891-Glu and Ile-4734 Multiple Mutations of Ryanodine Receptor in the Fall Armyworm, Spodoptera frugiperda.

The key mutations, such as the Gly-4891-Glu substitution and the Ile-4734 multiple substitutions within the ryanodine receptors (RyR), are linked to diamide resistance in fall armyworm (FAW), Spodoptera frugiperda. In this study, we found that FAW remained sensitive to cyantraniliprole and chlorantraniliprole, while its sensitivity to flubendiamide was reduced. Moreover, a low level of heterozygous mutation at I4743 was observed. To facilitate the detection procedure of these mutations, a simple and efficient loop-mediated isothermal amplification (LAMP) protocol was developed for operation. The reaction for detecting the G4891E and I4743 single or multiple mutations was carried out at 68 °C for 85 min and 68 °C for 85 min or 68 °C for 65 min, respectively. These LAMP reactions can be easily observed via visualization of the color change from pink to yellow. This assay provides a simple, convenient, and effective means of detecting mutations in the RyR of FAW for pest management purposes.

Loop-mediated isothermal amplification assay detects multiple alleles of bla OXA-51-like genes in Acinetobacter baumannii and other Gram-negative bacteria despite primer-template mismatches.

The known intrinsic and polymorphic bla OXA-51-like genes of Acinetobacter baumannii were recently reported in other non-A. baumannii Gram-negative pathogens. Accurate detection of this potentially transferrable carbapenemase gene in the clinical setting is critical. This study developed a loop-mediated isothermal amplification (LAMP) assay targetting multiple alleles of bla OXA-51-like genes. Specifically, an alignment-based primer design, in silico primer screening, and in vitro assay confirmation were conducted. Both in silico and in vitro results revealed the tolerance of the LAMP assay to up to five primer-template mismatches outside the 3'-end primer regions. Within 90 min, the LAMP assay also detected the gene targets in other Gram-negative bacteria with known and novel bla OXA-51-like genes. Finally, it showed a superior limit of detection (as low as 101 CFU/mL) compared with polymerase chain reaction, and high specificity against non-targets. This study developed a highly adaptable LAMP assay to monitor bla OXA-51-like genes in the clinical setting and provided important insights into LAMP primer design and screening.

Development and evaluation of rapid and simple detection of Klebsiella pneumoniae using closed dumbbell-mediated isothermal amplification diagnostic assay.

Introduction: Klebsiella pneumoniae (K. pneumoniae) is the most common pathogen causing hospital respiratory tract infection and epidemic. Gold standard procedures of microscopic examination and biochemical identification are widely used in clinical diagnosis with disadvantages of low sensitivity, time-consuming and sophisticated equipment requiring. An efficient, nucleic acid amplification-based sensitive and specific on-site identification of K. pneumoniae in clinical is necessary to facilitate clinical medication and disease control. Methods: We developed a closed dumbbell mediated isothermal amplification (CDA) assay for the rapid and sensitive detection of conserved rcsA gene in K. pneumoniae by real-time fluorescence monitoring and end-point colorimetric judgement. We designed and selected a pair of inner primers of CDA to detect K. pneumoniae. Then outer and loop primers were designed and verified to accelerate CDA reaction to achieve more efficient detection of K. pneumoniae. Results: The results showed the detection limit of CDA assay was 1.2 × 10-5 ng/µL (approximately 1 copy of the target gene) within 60 min, which was 100-fold more sensitive than real-time quantitative PCR (qPCR). Several pathogen genomic DNAs (Staphylococcus aureus, Shigella sonnei, Vibrio parahaemolyticus, Escherichia coli, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida albicans, Streptococcus agalactiae, Rickettsia, Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella oxytoca, and Klebsiella aerogenes) were used to evaluate the sensitivity and specificity of the established K. pneumoniae CDA assay. Total 224 batches of samples from other strains tested were negative and 296 batches of extracted K. pneumoniae DNA samples were positive by the developed CDA amplification approach, revealing high specificity and specificity of the diagnostic assay. In addition, the results of real-time fluorescence amplification of the K. pneumoniae CDA were in consistent with those of end-point colorimetric results. Discussion: The established real-time fluorescence and visual CDA assays of K. pneumoniae with merits of rapid, sensitive and specificity could be helpful for on-site diagnosis and clinical screening in rural areas.

Rapid and simultaneous detection of common childhood diarrhea viruses by microfluidic-FEN1-assisted isothermal amplification with ultra-high specificity and sensitivity.

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea-rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/µL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis.

Isothermal Technologies for HPV Detection: Current Trends and Future Perspectives.

The human papillomavirus (HPV) is a non-enveloped DNA virus transmitted through skin-to-skin contact that infects epithelial and mucosal tissue. It has over 200 known genotypes, classified by their pathogenicity as high-risk and low-risk categories. High-risk HPV genotypes are associated with the development of different types of cancers, including cervical cancer, which is a leading cause of mortality in women. In clinical practice and the market, the principal tests used to detect HPV are based on cytology, hybrid detection, and qPCR. However, these methodologies may not be ideal for the required timely diagnosis. Tests have been developed based on isothermal nucleic acid amplification tests (INAATs) as alternatives. These tests offer multiple advantages over the qPCR, such as not requiring specialized laboratories, highly trained personnel, or expensive equipment like thermocyclers. This review analyzes the different INAATs applied for the detection of HPV, considering the specific characteristics of each test, including the HPV genotypes, gene target, the limit of detection (LOD), detection methods, and detection time. Additionally, we discuss the tests available on the market that are approved by the Food and Drug Administration (FDA). Finally, we address the challenges and potential solutions for the large-scale implementation of INAATs, particularly in rural or underserved areas.

Identification of Pathogen Causing Bulb Rot in Fritillaria taipaiensis P. Y. Li and Establishment of Detection Methods.

Fritillaria taipaiensis P. Y. Li (F. taipaiensis) is a traditional Chinese herbal medicine that has been used for over two millennia to treat cough and expectoration. However, the increasing cultivation of F. taipaiensis has led to the spread of bulb rot diseases. In this study, pathogens were isolated from rotten F. taipaiensis bulbs. Through molecular identification, pathogenicity testing, morphological assessment, and microscopy, Fusarium solani was identified as the pathogen causing bulb rot in F. taipaiensis. The colonization of F. solani in the bulbs was investigated through microscopic observation. The rapid and accurate detection of this pathogen will contribute to better disease monitoring and control. Loop-mediated isothermal amplification (LAMP) and qPCR methods were established to quickly and specifically identify this pathogen. These results provide valuable insights for further research on the prediction, rapid detection, and effective prevention and control of bulb rot in F. taipaiensis.

Visualized detection of tobacco anthracnose by RPA-LFD.

Anthracnose caused by Colletotrichum spp. is a widespread fungal disease that is detrimental to tobacco growth and inflicts economic damage up to 100 million in tobacco-growing regions in China. An early diagnostic tool is vital for the accurate determination and management of anthracnose in the field. This study investigated the diversity of Colletotrichum spp. on tobacco leaves with anthracnose and developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) diagnostic method for the rapid and equipment-independent detection of the main Colletotrichum spp. causing tobacco anthracnose. This assay targeted the chitin synthase gene (chs1) and could be performed in a few minutes (6-10 min). All isolates of C. kastii, C. fructicola and C. gloeosporioides yielded positive results using the RPA-LFD assay, and no cross-reaction occurred with other fungal species from tobacco or other hosts. The detection threshold was 1 pg of genomic DNA under optimal reaction conditions. The entire RPA-LFD assay enabled the detection of pathogen visualization within 30 min without specialized equipment by combining a polyethylene glycol-KOH method for extracting DNA rapidly from tobacco leaves infected with C. kastii, C. fructicola and C. gloeosporioides. Based on these results, the RPA-LFD assay is easy to operate, rapid and equipment independent and is promising for development as a kit to diagnose tobacco anthracnose in resource-limited settings at point-of-care.

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10 nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

Colorimetric Loop-Mediated Isothermal Amplification Assays Accurately Detect blaOXA-23-like and ISAba1 Genes from Acinetobacter baumannii in Pure Cultures and Spiked Human Sera.

Carbapenem resistance in Acinetobacter baumannii is a critical global health threat attributed to transferrable carbapenemase genes. Carbapenemase genotyping using polymerase chain reaction (PCR) presents a challenge in resource-limited settings because of its technical requirements. This study designed new loop-mediated isothermal amplification (LAMP) primers using multiple sequence alignment-based workflows, validated the primer performance against multiple target variants in silico, and developed novel LAMP assays (LAntRN-OXA23 and LAntRN-ISAba1) to detect the transferable blaOXA-23-like carbapenemase genes and ISAba1 elements in pure cultures and A. baumannii-spiked serum samples. The designed LAMP primers bind to the conserved regions of their highly polymorphic targets, with their in silico performance comparable with other published primers. The in vitro LAMP assays (using 30 PCR-profiled A. baumannii and 10 standard multidrug-resistant gram-negative isolates) have 100% concordance with the PCR-positive clinical samples, limits of detection as low as 1 pg/µL (200 copies/µL), and specificities of 57.89-100%. Both assays produced positive results when testing DNA samples (extracted using a commercial kit) from blaOXA-23-like and ISAba1-blaOXA-51-like PCR-positive A. baumannii-spiked normal human sera (five set-ups per target). In summary, the LAMP assays accurately detected the target genes and have applications in infection management, control, and point-of-care testing in resource-limited healthcare settings.

An Integrated Micro-Heating System for On-Chip Isothermal Amplification of African Swine Fever Virus Genes.

The loop-mediated isothermal amplification (LAMP) is widely used in the laboratory to facilitate rapid DNA or RNA detection with a streamlined operational process, whose properties are greatly dependent on the uniformity and rise rate of temperature in the reaction chambers and the design of the primers. This paper introduces a planar micro-heater equipped with an embedded micro-temperature sensor to realize temperature tunability at a low energy cost. Moreover, a control system, based on the Wheatstone bridge and proportional, integral, and derivative (PID) control, is designed to measure and adjust the temperature of the micro-heater. The maximum temperature rise rate of the designed micro-heater is ≈8 °C s-1, and it only takes ≈60 s to reach the target temperature. Furthermore, a designed plasmid, containing the B646L gene of African Swine Fever Virus (ASFV), and a set of specific primers, are used to combine with the designed micro-heating system to implement the LAMP reaction. Finally, the lateral flow assay is used to interpret the amplification results visually. This method can achieve highly sensitive and efficient detection of ASFV within 40 min. The sensitivity of this on-chip gene detection method is 8.4 copies per reaction, holding great potential for applications in DNA and RNA amplification.

Advancements and applications of loop-mediated isothermal amplification technology: a comprehensive overview.

Loop-mediated isothermal amplification (LAMP) is a novel method for nucleic acid detection known for its isothermal properties, high efficiency, sensitivity, and specificity. LAMP employs 4 to 6 primers targeting 6 to 8 regions of the desired sequence, allowing for amplification at temperatures between 60 and 65°C and the production of up to 109 copies within a single hour. The product can be monitored by various methods such as turbidimetry, fluorometry, and colorimetry. However, it faces limitations such as the risk of non-specific amplification, challenges in primer design, unsuitability for short gene sequences, and difficulty in multiplexing. Recent advancements in polymerase and primer design have enhanced the speed and convenience of the LAMP reaction. Additionally, integrating LAMP with technologies like rolling circle amplification (RCA), recombinase polymerase amplification (RPA), and CRISPR-Cas systems has enhanced its efficiency. The combination of LAMP with various biosensors has enabled real-time analysis, broadening its application in point-of-care testing (POCT). Microfluidic technology has further facilitated the automation and miniaturization of LAMP assays, allowing for the simultaneous detection of multiple targets and preventing contamination. This review highlights advancements in LAMP, focusing on primer design, polymerase engineering, and its integration with other technologies. Continuous improvements and integration of LAMP with complementary technologies have significantly enhanced its diagnostic capabilities, making it a robust tool for rapid, sensitive, and specific nucleic acid detection with promising implications for healthcare, agriculture, and environmental monitoring.

Field-applicable simultaneous multiplex LAMP assay for screening HBV and HCV co-infection in a single tube.

BACKGROUND: Globally, around 7 to 20 million people are believed to be suffering from coinfection with both hepatitis B virus (HBV) and hepatitis C virus (HCV). The loop-mediated isothermal amplification (LAMP) approach, introduced by Notomi and colleagues, has undergone substantial advancements as an effective molecular tool that enables the simultaneous analysis of multiple samples in a single tube. METHODS: The present study examined the simultaneous detection of HBV and HCV in a single tube using melt curve analysis multiplex LAMP (mLAMP), which is based on the identification of unique melting peak temperatures. Selected regions for primer design including the S gene of HBV and the UTR gene of HCV. Primer optimization is initially performed through individual HBV and HCV LAMP analysis. Following the optimization process, the mLAMP assay was evaluated by optimizing the multiplex reaction mixture, determining the reaction time, and analyzing the limit of detection (LOD). The results are also analyzed using lateral flow dipsticks (LFD), which enable the visual detection of HBV and HCV by adding 20 pmol FITC-labeled LF primers into the reaction mixture prior the mLAMP. RESULTS: The LOD for the mLAMP assay was determined as 10 copies/µl, and no cross-reactivity with other microorganisms was detected. The detection results obtained from patient plasma were also visually demonstrated using LFD, and displayed significant concordance with those obtained from Real-Time Polymerase Chain Assay. The mLAMP assay revealed a diagnostic sensitivity of 95% for detecting the HBV, and LOD is 90% for HCV. The overall diagnostic sensitivity of the mLAMP assay for both viruses was 85%. The assay confirmed a specificity of 100%. CONCLUSION: The mLAMP assay displays significant promise for analyzing coinfected samples by simultaneously detecting the dual targets HBV and HCV within a set temperature of 62 °C, all within a time frame of 1 h. Additionally, when paired with disposable LFD, the mLAMP assay enables rapid visual detection of assay results in a matter of minutes. The result contributes to the mLAMP assay being highly suitable for coinfection screening, particularly in field conditions.

Detection and differentiation of low virulence and virulent Orthoavulavirus javaense using a molecular beacon with RT-LAMP.

Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (104 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RT-LAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.