Loop-mediated Isothermal Amplification Method Research Articles

Loop-mediated isothermal amplification assay coupled with lateral flow dipstick for the rapid detection of methicillin-resistant Staphylococcus pseudintermedius from dogs.

Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.

Loop-Mediated Isothermal Amplification-Based Workflow for the Detection and Serotyping of Salmonella spp. in Environmental Poultry Flock Samples.

Salmonella spp. is one of the most important foodborne pathogens worldwide. Given the fact that poultry and poultry products are the main source of human infection, Salmonella control in these farms is of utmost importance. To better control this pathogen in farms, boot swabs are used to sample farm environments but the analysis of these swabs is mainly based on culture-dependent methods. In the present study, a novel loop-mediated isothermal amplification (LAMP) method was developed for the rapid screening of Salmonella spp. in boot swab samples from broiler flock environments. Four different DNA extraction protocols were evaluated in depth, including a simple thermal lysis, a chelex-based protocol and two thermal lysis protocols followed by the purification of magnetic beads made of silica ("glass milk") in order to determine the most suitable alternative for potential on-site, farm analyses. The methodology evaluation included a blind interlaboratory assay and as a proof-of-concept, a naked-eye colorimetric assay was also included. Following the final methodology, it was possible to reach an LoD50 of 1.8 CFU/25 g of the samples, with a high relative sensitivity (95.7%), specificity (100%) and accuracy (96.6%) along with Cohen's kappa of concordance with respect to the ISO standard 6579-1:2017 of 0.9, with an RLOD of 1.3. In addition to this, due to the relevance of certain serotypes with the genus Salmonella spp., a serotype LAMP panel for the specific identification of S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar and S. Virchow was also developed. Even though some degree of cross-reactivity among the primers developed was observed, all the serotypes could be accurately identified based on their melt curve analysis profile. Taken together, in the present study, a rapid Salmonella spp. screening method, suitable for farm applications, was developed, along with a serotyping panel that could be used in a laboratory setup for the identification of the most relevant serotypes of the genus, taking advantage of real-time amplification followed by melt curve analysis.

Prospective evaluation of non-invasive saliva specimens for the diagnosis of syphilis and molecular surveillance of Treponema pallidum.

The promising diagnostic performance of molecular testing for syphilis using saliva and urine samples has been reported; however, further evaluation of its possible application for diagnosis and molecular surveillance is required. In addition, the development of a rapid and easy-to-perform molecular test for syphilis is important for its use in the clinical setting. We comprehensively evaluated the diagnostic and surveillance performance of two novel loop-mediated isothermal amplification (LAMP) assays using saliva and urine samples. Saliva, urine, and whole blood were collected from patients who underwent serological testing for syphilis at outpatient clinics. Treponema pallidum DNA in specimens was detected using quantitative PCR (qPCR), nested PCR, and novel LAMP assays. T. pallidum genotyping was conducted by multi-locus sequence typing (MLST). Of the 163 patients recruited, 98 were diagnosed with syphilis (primary: n = 35; secondary: n = 40; latent: n = 23). qPCR showed the highest sensitivity among the molecular tests performed with a sensitivity of 54.1% and 30.3% for all syphilis patients using saliva and urine samples, respectively. A novel method of LAMP combined with dry reagents and crude DNA extraction (Dry-LAMP) showed a probit detection limit of 37.4 copies/reaction within 45 min. The agreement rate between Dry-LAMP and qPCR for saliva was 95.7% (κ coefficient 0.90). The T. pallidum genotype was identified in 48 patients by MLST using saliva samples. Molecular analysis of saliva could be used as a supplementary diagnostic test for syphilis and molecular surveillance of the T. pallidum genotype. Dry-LAMP is expected to be helpful in the clinical diagnosis of syphilis.

Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry.

Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world's most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions) and designing loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani. Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with quadplex, triplex, and singleplex, respectively. None of the assays were impacted by any inhibitory or competitive effects after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 9 min. The reproducibility was confirmed by performing the assay in two independent laboratories. These assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection, disease diagnosis, and epidemiological studies.IMPORTANCEDickeya solani, one of the most aggressive soft rot causing bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to the potato industry. Accurate and timely detection of this bacterium is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research, we have developed LAMP and multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani. These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool enabling management of this threatening pathogen.

Application of Loop-Mediated Isothermal Amplification for Edwardsiella Ictaluri Detection in Indonesia Enteric Septicemia of Catfish

Abstract Loop-mediated isothermal amplification (LAMP) method is useful for rapidly detecting Edwardsiella ictalurid infection, especially enteric septicemia of catfish (ESC). This study aims to investigate the LAMP method for E. ictaluri detection in ESC. This research was an experimental study using a total of 55 catfish, consisting of 5 negative controls and 50 catfish injected intraperitoneally with 0.1 mL of E. ictaluri at a concentration of 105 CFU/mL. The kidneys of three fishes were randomly selected 6, 12, 18, 24, 30, 36, 42, 48, and 54 hours after infection with E. ictaluri. In addition, samples were also collected on days 3, 5, and 7. Bacterial analysis was determined by conventional biochemistry (genus and species test), whole-genome sequencing of catfish, and LAMP amplification. All types of data obtained in this study were analyzed by descriptive statistics to compare infected and healthy catfish. The catfish were infected with E. ictaluri after 12 hours of infection according to the LAMP amplification procedure which revealed hemorrhages throughout the body, fins, protruding eyes, necrosis, inflammation of the spleen, liver, intestine, pale gills, gut, abdomen swelling, and tissue necrosis in the upper part of the head. It can be concluded that the LAMP method is more effective than the PCR method for detecting infection with E. ictaluri in catfish.

Development of a loop-mediated isothermal amplification method for the rapid detection of Clostridium botulinum serotypes E and F.

Botulinum neurotoxin serotypes E and F (BoNT/E and BoNT/F) produced by the bacteria Clostridium botulinum (C. botulinum) found in a wide variety of foods cause poisoning in humans with high mortality rates. Mouse bioassays (MBAs), the gold standard method for BoNT detection, have a low detection limit; however, require experienced personnel and take a long time to obtain results. Therefore, it has been gradually replaced by nucleic acid amplification tests (NAATs) with primers targeting species-specific genes. In this study, for each serotype E and F, six LAMP primers were designed based on multiple sequence alignments of the conserved regions of the bont/E and bont/F genes collected from 180 serotype E strains and 23 serotype F strains published in NCBI. In silico PCR amplification with the outer primer pairs showed successful amplification of the target fragments. To validate the LAMP method, we constructed two synthetic plasmids containing the target sequences extended approximately 10-50bp to both ends. The specificity of the primers was further evaluated using six different Clostridium species and eight strains belonging to other common food poisoning-related bacterial species. Employing the synthetic plasmids, the optimal temperatures and limits of detection (LODs) were determined for bont/E (63°C, LOD ≤ 101 copies/reaction) and bont/F (65°C, LOD ≤ 102 copies/reaction) within 30min. In addition, the LAMP primer set for BoNT/F was redesigned with degenerate nucleotides that improved the coverage from 15 to 45%. For future directions, applications of the established method, especially with the degenerate primers, could be used as an alternative assay for the rapid and sensitive detection of C. botulinum.

Cloning and Optimized Expression of Bst DNA Polymerase from Geobacillus stearothermophillus in Escherichia coli BL21

Bst DNA polymerase possesses strand displacement activity, enabling isothermal DNA amplification without requiring a thermal cycler. This enzyme is utilized in the Loop-Mediated Isothermal Amplification (LAMP) method, which offers advantages in speed and simplicity over Polymerase Chain Reaction (PCR) method. The growing demand for Bst DNA polymerase highlights the need for cost-effective in-house production, as a commercial option is economically challenging. For that purpose, this study aims to construct and optimize the expression of Bst DNA polymerase from Geobacillus stearothermophilus in Escherichia coli. The expression constructs pET16b.BstHF vector was constructed using Gibson Assembly and expressed in E. coli BL21 (DE3). Optimal expression was achieved with 1 mM IPTG, induction at OD600 0.8 and 6-hour induction time. The purified enzyme was achieved with a protein yield of 2,175 mg/L culture and demonstrated effective polymerase activity for LAMP.

Detection of Vibrio harveyi through RPA combined with CRISPR/Cas12a: Construction of a new method and comparative study

In this study, recombinase polymerase isothermal amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas) to establish a new method (RPA-CRISPR/Cas12a) for the detection of Vibrio harveyi. We used RPA to amplify the target DNA of V. harveyi and the RPA amplification product as an activator to initiate CRISPR/Cas12a cleavage of the designed ssDNA reporter probe. Then, we used fluorescence intensity as a criterion for the detection results to optimize the reaction conditions and improve the accuracy of the assay. The ToxR-specific gene sequence of V. harveyi was selected, and 3 sets of RPA primers and 2 sets of gRNA were designed for screening to optimize the ratio of LbCas12a protein, gRNA, and reporter. The specificity, sensitivity and detection of the newly developed method on simulated contaminated samples were analyzed and compared with those of the RPA method, fluorescent loop mediated isothermal amplification (LAMP), and visual LAMP methods. The results showed that the newly constructed method exhibited good specificity and no cross-reactivity with other pathogenic bacteria. Maximum fluorescence values were obtained when the molar concentration ratios of LbCas12a protein to reporter and gRNA were 1:0.8 and 1:8, respectively. The sensitivities of RPA-CRISPR/Cas12a for the detection of V. harveyi were 10−5 ng/μL, respectively, and the sensitivities for the simulated contaminated samples were 7 CFU/mL, respectively. The new RPA-CRISPR/Cas12a method provides a new strategy for the immediate detection of foodborne pathogenic microorganisms.

Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid, Sensitive, and Specific Detection of Hazelnut as an Allergen in Food Matrix.

Analytical verification of hazelnut in food supports risk-based approaches for proper allergen labeling to prevent unwanted allergic reactions or to quality control diagnostic or therapeutic allergen preparations for allergic subjects. We present the development and validation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of hazelnut by targeting the internal transcribed spacer 2 gene. The qualitative method requires neither sophisticated analytical equipment nor antibodies, allowing an easy-to-use application with no ethical concern related to the use of animals. It demonstrated a limit of detection at or below 10 mg/kg hazelnut in various food matrices, making it also suitable for verifying hazelnut at levels of clinically relevant eliciting doses. Validation against proficiency test samples and testing of applicability with commercial food items confirmed its usefulness in processed foods. The simplicity of the method, including visual colorimetric detection, combined with high specificity and sensitivity, represents an advancement over existing qualitative methods.

Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.

A colorimetric reverse-transcription loop-mediated isothermal amplification method targeting the L452R mutation to detect the Delta variant of SARS-CoV-2

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay’s 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.

Development of machine learning algorithm for loop-mediated isothermal amplification including influence of temperature

To predict amplification in the Loop-Mediated Isothermal Amplification (LAMP) method, a binary model has been developed. This model assesses whether the combination of partial template binding facilitated by multiple primers aligns with a specific signature indicating amplification. For this model, 1512 settings were adjusted, encompassing variations in the extraction oligomer position from the primer, the oligomer length, the number of allowed mismatched bases, and the stringency of the homology position. The optimal set was determined based on the F1 value at a reaction temperature of 60°C, resulting in a value of 0.58. To implement machine learning by amalgamating multiple models, duplicate models were eliminated through cluster analysis. Machine learning using AutoML was performed on 108 distinct models. Consequently, the F1 values were 0.7119 at a reaction temperature of 60°C and 0.7210 at 68°C. The weak correlation between feature importance and the F1 value of the model alone suggests that machine learning utilizing this model incorporates numerous models to enhance prediction accuracy.

Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for Candida glabrata detection.

Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata. The specific LAMP primers for C. glabrata detection were designed and evaluated. The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species. The developed molecular method would be a promising tool in the management of invasive candidiasis.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

A colorimetric assay for Neospora caninum utilizing the loop-mediated isothermal amplification technique

Neospora caninum (N. caninum) is a protozoan parasite that poses a serious risk to livestock by infecting various domestic and wild animals. Loop-Mediated Isothermal Amplification (LAMP) offers a cost-effective, highly sensitive, and specific method for detecting protozoan parasites. This study aims to develop a precise, rapid, and visually assessable colorimetric LAMP method, improving on traditional techniques. We employed a rigorous screening process to identify the optimal primer set for this experiment. Subsequently, we fine-tuned the LAMP reaction at 65 °C for 40 min with 270 μmol/L neutral red. We then confirmed the specificity of primers for N. caninum through experimental validation. The LAMP method demonstrated a lower detection limit compared to traditional Polymerase Chain Reaction (PCR) techniques. While LAMP offers clear advantages, the prevalence of DNA detected in 89 sheep serum and 59 bovine serum samples using the nested PCR method was 3.37 % (3/89) and 1.69 % (1/59), respectively. In contrast, when the LAMP method was employed, the prevalence of detected DNA rose to 5.61 % (5/89) for sheep and 3.38 % (2 /59) for bovine. A comparison of two molecular assays using the intragroup correlation coefficient (ICC) resulted in a value of 0.999 (95 % CI: 0.993–0.996, p < 0.001), indicating the LAMP method is in the “better” range according to James Lee's categorization. The LAMP technique, optimized with specific primers of N. caninum and neutral red dye, not only exhibited higher sensitivity but also provided convenience over conventional PCR methods, highlighting its potential for on-site applications and cost-effective field detection.

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.

Molecular Detection of Respiratory Tract Viruses in Chickens at the Point of Need by Loop-Mediated Isothermal Amplification (LAMP).

Accurate and timely molecular diagnosis of respiratory diseases in chickens is essential for implementing effective control measures, preventing the spread of diseases within poultry flocks, minimizing economic loss, and guarding food security. Traditional molecular diagnostic methods like polymerase chain reaction (PCR) require expensive equipment and trained personnel, limiting their use to centralized labs with a significant delay between sample collection and results. Loop-mediated isothermal amplification (LAMP) of nucleic acids offers an attractive alternative for detecting respiratory viruses in broiler chickens with sensitivity comparable to that of PCR. LAMP's main advantages over PCR are its constant incubation temperature (∼65 °C), high amplification efficiency, and contaminant tolerance, which reduce equipment complexity, cost, and power consumption and enable instrument-free tests. This review highlights effective LAMP methods and variants that have been developed for detecting respiratory viruses in chickens at the point of need.

Rapid cyprinid herpesvirus 3 detection using loop-mediated isothermal amplification (LAMP) combined with gold nanoprobes and SYBR safe

As an etiological agent of a notifiable and emerging disease, Cyprinid herpesvirus 3 (CyHV-3 or koi herpesvirus, KHV) is a highly risky factor that affects koi and common carp yield through increasing the mortality rates. In the current study, a highly rapid and sensitive technique was established to detect CyHV-3 with the help of loop-mediated isothermal amplification (LAMP) method with dual visualizing indicators (SYBR safe and gold nanoparticle probes; AuNP-probes). Six specific primers were used to amplify the thymidine kinase (TK) of CyHV-3 using a LAMP reaction of 45 min at 65 °C along with detecting the products through visual inspection via hybridization at 65 °C for 20 min with a functionalized thiol-AuNP probe. A closed-tube LAMP-SYBR safe assay was also developed under the same condition without AuNP probe. The amplification of KHV-LAMP products were evaluated by the naked eye, through fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric aggregate of LAMP product in the presence of AuNP probe. Furthermore, quantitative measurement was applied to the LAMP-AuNPs assay, using the spectral shift assay. The detection limit was 1.25 fentograms DNA which can compete the latest techniques available. The proposed technique yielded negative outcomes with DNA template from other viruses such as CyHV-1 and CEV. The LAMP-SYBR safe and LAMP-AuNPs assays introduced in this article were very sensitive, fast, and reliable diagnostic tools for the detection of CyHV-3.

Multiplex LAMP assay for detecting the prevalent species of dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus in the domestic environment

Dermatophagoides farina (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus) are the prevalent kinds of house dust mites (HDMs). HDMs are common inhalant allergens that cause a range of allergic diseases, such as rhinitis, atopic dermatitis, and asthma. The epidemiology of these diseases is associated with exposure to mites. Therefore, in the present study, a method named multiplex loop-mediated isothermal amplification (LAMP) was developed to detect environmental dust mites. The multiplex LAMP assay allows amplification within a single tube and has an ITS plasmid detection limit as low as 40 fg/µL for both single dust mites and mixed dust mites (D. pteronyssinus and D. farinae), which is up to ten times more sensitive than classical PCR techniques. Furthermore, the multiplex LAMP method was applied to samples of single dust mites and clinical dust to confirm its validity. The multiplex LAMP assay exhibited higher sensitivity, simpler instrumentation, and visualization of test results, indicating that this method could be used as an alternative to traditional techniques for the detection of HDMs.