Sensitivity Of Loop-mediated Isothermal Amplification Research Articles

Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, 4 μg/μL of myoglobin, 10 μg/μL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

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

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

Rapid and quantitative detection of Aspergillus niger Van Tieghem using loop-mediated isothermal amplification assay

AbstractPeanut (Arachis hypogaea L.) crown rot and root rot are common diseases caused by Aspergillus niger Van Tieghem. Early and accurate detection of A. niger is key to disease management. In this study, the design of two to five sets of loop-mediated isothermal amplification (LAMP) primers was based on the EglA, GOD, Tub, NRPS, Tan, CbhA, and CbhB genes of A. niger. Of these, primer set GOD-91 was selected for optimization of the three-factor LAMP system: the Bst DNA polymerase concentration, the concentration ratio of the inner and outer primers, and the concentration of Mg2+. In addition, the optimized LAMP reaction system for A. niger detection was validated for specificity, sensitivity, and on-site feasibility. The specificity test showed that A. niger could be specifically detected with the proposed method without cross-amplification of other pathogenic fungi DNA. Moreover, based on the sensitivity test, the lowest detection limit of this reaction system was 5.1 × 10−7 ng/µL pAN01 plasmid DNA, after which a standard curve was generated for the quantitative detection of A. niger. The LAMP method was further applied for field sample assessment before and after A. niger infection, successfully detecting A. niger presence in the samples collected in the field. This study yielded a sensitive, specific, and reproducible LAMP system that can be used to assess on-site samples within 45 min. It is an effective approach for the rapid and quantitative detection of A. niger.

Application of LAMP assay for detection of carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex in ICU admitted sepsis patients: A rapid and cost-effective diagnostic tool

Carbapenem-resistant significant members of Acinetobacter calcoaceticus–Acinetobacter baumannii (CR-SM-ACB) complex have emerged as an important cause of sepsis, especially in ICUs. This study demonstrates the application of loop-mediated-isothermal-amplification (LAMP) assay for detection of CR-SM-ACB-complex from patients with sepsis.Whole-blood and culture-broths(CB) collected from patients with culture-positive sepsis were subjected to LAMP and compared with PCR, and RealAmp. Vitek-2 system and conventional PCR results were used as confirmatory references.The sensitivity and specificity of LAMP(97 % & 100 %) and RealAmp(100 % & 100 %) for detection of CR-SM-ACB-complex from CB were better than PCR(87 % & 100 %). Diagnostic accuracy of LAMP, RealAmp, and PCR for detection of SM-ACB-complex from CB was 98.5 %, 100 %, and 88.5 % respectively. Turnaround time of Culture, LAMP, PCR, and RealAmp was 28-53, 6-20, 9-23, and 6-20hours, respectively.LAMP is a simple, inexpensive tool that can be applied directly to positive CB and may be customized to detect emerging pathogens and locally-prevalent resistance genes and to optimize antimicrobial use.

Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94fg/µLand 25.8fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12h enrichment. The study highlights the importance of short (6-12h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus

BackgroundThe monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. ResultsHere, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SignificanceThe device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

LAMP Assay to Detect Elsinoë necatrix, an Important Eucalyptus Shoot and Leaf Pathogen.

Eucalyptus scab and shoot malformation caused by Elsinoë necatrix is an emerging disease and a serious threat to the global commercial forestry industry. The disease was first discovered in North Sumatra, Indonesia, and now requires a simple and effective method for early pathogen detection. In this study, a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for E. necatrix. A unique region in a secondary metabolite gene cluster was used as a target for the assay. To test robustness of the assay, LAMP was verified in 15 strains of E. necatrix. A specificity test against 23 closely related Elsinoë species and three fungal species commonly isolated on Eucalyptus showed that the LAMP assay exclusively identified E. necatrix isolates. The assay had a high level of sensitivity, able to detect 0.01 ng (approximately 400 target copies) of pure E. necatrix DNA. Furthermore, using a simple DNA extraction method, it was possible to use this assay to detect E. necatrix in infected Eucalyptus leaves.

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

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

A handheld isothermal fluorescence detector for duplex visualization of aquatic pathogens via enhanced one-pot LAMP-PfAgo assay

The expansion of large-scale aquaculture has exacerbated the challenge of aquatic diseases, resulting in substantial economic losses annually. Currently, traditional laboratory-based diagnostic methods are time-consuming and costly, hindering on-site testing for individual farmers. We address this issue by developing a state-of-the-art handheld isothermal nucleic acid amplification device (WeD-1) capable of fluorescence tracking of reactions and integrating it with an enhanced one-pot Prokaryotic Argonaute based nucleic acid detection method, enabling duplex visual detection of aquatic pathogens. WeD-1 is portable, reusable, user-friendly, and cost-effective, offering real-time smartphone interaction and enabling real-time fluorescence observation during the reaction. The enhanced one-pot Loop-Mediated Isothermal Amplification (LAMP)-PfAgo method, incorporating paraffin-encapsulated lyophilized PfAgo protein, achieves precise target-specific cleavage, significantly enhancing multiplex nucleic acid detection. This innovation streamlines on-site testing, negating the need for specialized laboratory conditions while ensuring an aerosol-free system. With newly developed and highly sensitive LAMP primer sets, our compact WeD-1/LAMP-PfAgo nucleic acid rapid testing system exhibits remarkable sensitivity, readily detecting aquatic pathogens with naked eyes from rapidly prepared fish and shrimp samples within 40 min, even when the Ct values are as high as 34.

Evaluation of LAMP for Fasciola hepatica detection from faecal samples of experimentally and naturally infected cattle

Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a ‘rigorous’ FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a ‘rigorous’ FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture.

High-throughput characterization and phenotyping of resistance and tolerance to virus infection in sweetpotato

Breeders have made important efforts to develop genotypes able to resist virus attacks in sweetpotato, a major crop providing food security and poverty alleviation to smallholder farmers in many regions of Sub-Saharan Africa, Asia and Latin America. However, a lack of accurate objective quantitative methods for this selection target in sweetpotato prevents a consistent and extensive assessment of large breeding populations. In this study, an approach to characterize and classify resistance in sweetpotato was established by assessing total yield loss and virus load after the infection of the three most common viruses (SPFMV, SPCSV, SPLCV). Twelve sweetpotato genotypes with contrasting reactions to virus infection were grown in the field under three different treatments: pre-infected by the three viruses, un-infected and protected from re-infection, and un-infected but exposed to natural infection. Virus loads were assessed using ELISA, (RT-)qPCR, and loop-mediated isothermal amplification (LAMP) methods, and also through multispectral reflectance and canopy temperature collected using an unmanned aerial vehicle. Total yield reduction compared to control and the arithmetic sum of (RT-)qPCR relative expression ratios were used to classify genotypes into four categories: resistant, tolerant, susceptible, and sensitives. Using 14 remote sensing predictors, machine learning algorithms were trained to classify all plots under the said categories. The study found that remotely sensed predictors were effective in discriminating the different virus response categories. The results suggest that using machine learning and remotely sensed data, further complemented by fast and sensitive LAMP assays to confirm results of predicted classifications could be used as a high throughput approach to support virus resistance phenotyping in sweetpotato breeding.

Boron nitride nanoplate-based improvement of the specificity and sensitivity in loop-mediated isothermal amplification for Vibrio parahaemolyticus detection

BackgroundNucleic acid testing based on DNA amplification is gradually entering people's modern life for clinical diagnosis, food safety monitoring and infectious disease prevention. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are the most powerful techniques that have been the gold standard for quantitative nucleic acid analysis. However, the high nonspecific amplification rate caused by the formation of primer dimers, hairpin structures and mismatched hybridization severely restricts their real-world applications. It is highly desirable to explore a way for improving the specificity and sensitivity of PCR and LAMP assays. ResultsIn this work, we demonstrated that a nanomaterial boron nitride nanoplate (BNNP), due to its unique surface properties, can interact with the main components of the amplification reaction, such as single stranded primers and Bst DNA polymerase, and increase the thermal conductivity of the solution. As a result, the presence of BNNPs dramatically improved the specificity of PCR and LAMP. And BNNPs maintained the specificity even after five rounds of PCR. Moreover, the sensitivity of LAMP was also enhanced by BNNPs, and the detection limit of BNNP-based LAMP was two orders of magnitude lower than that of classical LAMP. Then the BNNP-based LAMP was applied to detect Vibrio parahaemolyticus in contaminated seafood samples with high specificity and a 10-fold increase in sensitivity. SignificanceThis is the first systematic demonstration of BNNPs as a promising additive to enhance the efficiency and fidelity of PCR and LAMP amplification reactions, thereby greatly expanding the application of nucleic acid detection in a wide range of laboratory and clinical settings.

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent.

Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma. We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas. In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.

Utility of a loop-mediated isothermal amplification detection kit to diagnose chronic pulmonary aspergillosis

BackgroundDiagnosing chronic pulmonary aspergillosis (CPA) is still challenging, and infection with Aspergillus spp. must be detected early. The aim of this study was to evaluate, for the first time, the usefulness of a new loop-mediated isothermal amplification (LAMP) test kit in diagnosing CPA. MethodsThis cross-sectional study was performed at the National Hospital Organization Kinki-Chuo Chest Medical Center. We prospectively enrolled patients with and without CPA. The primary outcomes of this study were the sensitivity and specificity of LAMP for the diagnosis of CPA. ResultsIn total, 34 patients with CPA and 21 without CPA (the control group) were enrolled between October 2019 and December 2021. Among the 34 patients with CPA, cultures were positive for CPA in 16 and negative for 18. The majority of fungi isolated in sputum were A. fumigatus in 15 specimens; Aspergillus flavus was isolated in 1) LAMP demonstrated 55.9% sensitivity (95% confidence interval [CI] 37.9–72.8) and 100.0% specificity (95% CI 77.2–100.0). ConclusionLAMP is an acceptable tool for diagnosing CPA, and its application in clinical practice necessitates more studies.

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

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

Detection of chronic toxoplasmosis in the brain of mice using loop-mediated isothermal amplification (LAMP) and conventional PCR

BackgroundToxoplasma gondii is a common protozoan parasite that infects approximately one-third of the world's population. It is a disease with multiple manifestations. In immunocompetent individuals, symptoms are mild and flu-like, whereas, in immunocompromised patients, it often results in severe morbidity and mortality. Thus, studies for developing a simple, rapid diagnostic tool for early detection of Toxoplasma are emerging. Molecular diagnosis is highly accurate and helpful in congenitally infected and immunocompromised patients. The loop-mediated isothermal amplification (LAMP) technique was invented to improve nucleic acid amplification efficacy in terms of sensitivity and specificity. Aim of the studyThe study aimed to validate a LAMP protocol for detecting Toxoplasma DNA in the brain homogenates from mice experimentally infected with Toxoplasma's ME-49 (cyst-forming type II) strain in comparison to PCR. MethodsIn this study, the target DNA fragment was the Toxoplasma 529-bp, repeated 200–300 copies/genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in brain homogenates in experimental mice at eight weeks post-infection and compared to the histopathology data. ResultsThe LAMP reaction showed positive results in 18 of the 26 examined samples of brain homogenates. PCR showed the characteristic 529-bp band in 15 of the 26 examined samples. ConclusionThe LAMP showed a higher sensitivity over PCR in detecting Toxoplasma infection in brain homogenates of infected mice.

Diagnostic utility of LAMP PCR targeting bcsp-31 gene for human brucellosis infection

PurposeHuman brucellosis is a neglected zoonotic disease of significant public health concern. Molecular diagnosis of brucella remains challenging in low resource settings, due to the high infrastructure and cost involved. Loop-mediated isothermal amplification (LAMP) is a rapid point of care polymerase chain reaction (PCR) with the utility of on-field molecular diagnosis and offers a convenient alternative to conventional PCR. In the present study, we developed and evaluated the diagnostic utility of in house LAMP PCR targeting the Brucella genus-specific bcsp-31 gene in patients having febrile illness. Materials and methodsThe analytical sensitivity and specificity of bcsp-31 LAMP PCR was first evaluated using brucella (n ​= ​8) and non-brucella cultures (n ​= ​5), along with spiked clinical samples. The overall diagnostic utility of developed LAMP PCR was then further evaluated in 393 human samples suspected of brucellosis. ResultsThe developed LAMP PCR could detect as low as 8 ​fg of DNA by visual detection within 35min. We report sensitivity and specificity of the developed LAMP PCR as 90.91% and 99.37%.The accuracy of the developed test assay was found to be 98.60%. In clinical samples, LAMP gave positivity of 20% with the concordance of 89% with conventional PCR. ConclusionTo conclude, a rapid, efficacious, sensitive LAMP PCR targeting the bcsp 31 gene was developed. The existing LAMP PCR can be used as a point of care screening test in various low resource endemic setting in lieu of conventional PCR for estimation of prevalence data, diagnosis and treatment of brucellosis.

Rapid point-of-care detection of BK virus in urine by an HFman probe-based loop-mediated isothermal amplification assay and a finger-driven microfluidic chip.

BK virus (BKV)-associated nephropathy (BKVN) is one of the leading causes of renal dysfunction and graft loss in renal transplant recipients. Early monitoring of BKV in urine is crucial to minimize the deleterious effects caused by this virus on preservation of graft function. We report a simple, rapid, sensitive loop-mediated isothermal amplification (LAMP) assay using an HFman probe for detecting BKV in urine. To evaluate the performance of the assay, a comparison of the HFman probe-based LAMP (HF-LAMP) assay with two qPCR assays was performed using urine samples from 132 HIV-1 infected individuals. We further evaluated the performance of HF-LAMP directly using the urine samples from these HIV-1 infected individuals and 30 kidney transplant recipients without DNA extraction. Furthermore, we combined the HF-LAMP assay with a portable finger-driven microfluidic chip for point-of-care testing (POCT). The assay has high specificity and sensitivity with a limit of detection (LOD) of 12 copies/reaction and can be completed within 30 min. When the DNA was extracted, the HF-LAMP assay showed an equivalent and potentially even higher sensitivity (93.5%) than the qPCR assays (74.2-87.1%) for 132 urine samples from HIV-1 infected individuals. The HF-LAMP assay can be applied in an extraction-free format and can be completed within 45 min using a simple heat block. Although some decreased performance was seen on urine samples from HIV-1 infected individuals, the sensitivity, specificity, and accuracy of the extraction-free BKV HF-LAMP assay were 95%, 100%, and 96.7% for 30 clinical urine samples from kidney transplant recipients, respectively. The assay has high specificity and sensitivity. Combined with a portable finger-driven microfluidic chip for easy detection, this method shows great potential for POCT detection of BKV.

Rapid Detection of A282S Mutation in the RDL1 Gene of Rice Stem Borer via the Mutation-Specific LAMP Technique

Rice stem borer Chilo suppressalis (Walker) is one of the most serious pests on rice and is distributed worldwide. With the long-term and continuous usage of insecticides, C. suppressalis has developed high levels of resistance to various kinds of insecticides, including phenylpyrazole insecticides. As is well known, the resistance of C. suppressalis to phenylpyrazole insecticides is determined by the A282S mutation of the GABA receptor RDL subunit. In order to efficiently detect the resistance of C. suppressalis, a rapid and sensitive loop-mediated isothermal amplification (LAMP) technique was established and optimized in this study. The optimal concentration of components was Bst DNA polymerase (0.24 U/μL), dNTP (0.8 mM), Mg2+ (4 mM), betaine (0.6 M), forward inner primer and backward inner primer (1.6 μM), F3 and B3 (0.4 μM), and hydroxyl naphthol blue (150 mM), respectively, and the optimal reaction condition was 63 °C for 60 min, which could reduce the cost and time of detection. In addition, the accuracy of the optimized LAMP reaction system and parameters was verified in the field strains of C. suppressalis from different regions, including Jiangsu, Jiangxi, and Hu’nan provinces. The mutation (A2’S) was successfully detected in the field strains. As far as we know, this is the first report of the LAMP technique applied in the resistance monitoring of C. suppressalis to phenylpyrazole insecticides. According to our results, the optimized LAMP reaction system is feasible and easy to operate and to efficiently detect resistance-related mutation in a short time, as directly judged by the naked eye. Our results provide a new tool for detection of resistance of C. suppressalis, which is a very useful tool for comprehensive management of C. suppressalis.

Analysis of the reliability of rapid diagnostic tests for varicella, including breakthrough cases.

In the era of universal varicella vaccination, diagnosis of varicella is challenging, especially for breakthrough cases. We sought to clarify the reliability of direct varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) and DermaQuick® VZV using the immunochromatography technique as rapid diagnostic tests for varicella. In addition, the usefulness of saliva as a sample type for direct LAMP was investigated. Among the 46 enrolled patients with suspected VZV infection, 31 patients (67.3%) were positive for the nucleic acid test based on real-time PCR from skin swab samples. Direct LAMP of skin swabs was positive in 29 (63.0%) of 46 patients. DermaQuick® VZV was positive in 25 (54.3%) of 46 patients. VZV DNA was detected in only 48.4% of oral swabs with the direct LAMP method. With real-time polymerase chain reaction (PCR) as the standard for diagnosing varicella, the sensitivity and specificity of DermaQuick® VZV were 80.7% and 100%, respectively. The sensitivity and specificity of direct LAMP from skin swabs were 93.6% and 100%, respectively. The sensitivity and specificity of real-time PCR for DNA extracted from oral swabs were 74.2% and 93.3%, respectively. Thus, oral swab samples are not suitable for breakthrough varicella diagnosis. Although DermaQuick® VZV is considered the most convenient point-of-care test for varicella, its sensitivity and specificity were lower than those of direct VZV LAMP.