Sensitivity Of Loop-mediated Isothermal Amplification Research Articles

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.

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.

Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.

Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S-23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63°C, and concentrations of the inner and loop primers were 1.4 and 1.0µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35min is 50pg, and at 60min it is 5fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.

Loop-mediated isothermal amplification (LAMP) test in the detection of uncomplicated malaria in pregnancy: a meta-analysis of diagnostic accuracy

BackgroundDue to relatively low malaria parasitaemia in pregnancy, an appropriate field test that can adequately detect infections in pregnant women presenting with illness or for malaria screening during antenatal care is crucially important. The objective was to evaluate the diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for the detection of uncomplicated malaria in pregnancy.MethodsThis was a meta-analysis of diagnostic accuracy. Relevant studies that assessed the diagnostic performance of LAMP for the detection of malaria in pregnancy were searched in health-related electronic databases including PubMed, Ovid, and Google Scholar. The methodological quality of the studies included was evaluated using the QUADAS-2 tool.ResultsOf the 372 studies identified, eight studies involving 2999 pregnant women in five endemic countries that assessed the accuracy of LAMP were identified. With three types of PCR as reference tests, the pooled sensitivity of LAMP was 91% (95%CI 67–98%) and pooled specificity was 99% (95%CI 83–100%, 4 studies), and the negative likelihood ratio was 9% (2–40%). Caution is needed in the interpretation as there was substantial between-study heterogeneity (I2: 80%), and a low probability that a person without infection is tested negative. With microscopy as a reference, the pooled sensitivity of LAMP was 95% (95%CI 26–100%) and pooled specificity was 100% (95%CI 94–100%, 4 studies). There was a wide range of sensitivity and substantial between-study heterogeneity (I2: 83.5–98.4%). To investigate the source of heterogeneity, a meta-regression analysis was performed with covariates. Of these potential confounding factors, reference test (p: 0.03) and study design (p:0.03) had affected the diagnostic accuracy of LAMP in malaria in pregnancy. Overall, there was a low certainty of the evidence in accuracy estimates.ConclusionThe findings suggest that LAMP is more sensitive than traditional tests used at facilities, but the utility of detecting and treating these low-density infections is not well understood. Due to the limited number of studies with bias in their methodological quality, variation in the study design, and different types of reference tests further research is likely to change the estimate. Well-conceived large prospective studies with blinding of the index test results are recommenced.

373. Development of multiplex loop-mediated isothermal amplification (LAMP) based on a microfluidic chip for detection of HSV-1 and 2 and VZV.

Abstract Background In the era of universal varicella vaccination, point-of-care testing for herpes simplex viruses 1 and 2 (HSV-1, HSV-2) and varicella-zoster virus (VZV) is becoming more important because of the increase in the number of breakthrough varicella cases. The Loop-mediated isothermal amplification (LAMP) method is a reliable method for rapid diagnostic tests for infectious diseases. Meanwhile, microfluidics technology is revolutionary in total system miniaturization, including facile parallelization through multiplexing and process-reduced reagent volumes. It is easy to create miniaturized chips containing multiple chambers according to the needs. In this study, we sought to develop multiplex LAMP for the detection of HSV-1, HSV-2, and VZV DNA using in-house microfluidic chips. Methods The polydimethylsiloxane-based chip consists of microfluidic channels and 5 reaction chambers. The specific primers were pre-spotted and dried in each chamber. The condition of LAMPs and each specific primer were described previously. The sensitivity of microfluidic LAMPs was confirmed using plasmids that contain the target sequences. In order to assess the reliability of clinical use, 35 swab samples (without DNA extraction) collected from patients with vesicular skin lesions were used. The result of microfluidic multiplex LAMP was compared with conventional LAMPs and real-time PCRs. Results Detection limits of HSV-1 and 2 and VZV LAMP-based on a microfluidic chip were 500 copies per reaction. Among 35 swab samples, HSV-1, HSV-2, and VZV DNA were detected in 10 (28.6%), 4 (11.4%), and 12 (34.3%) samples, respectively. The results of microfluidic LAMPs were completely consistent with the results of conventional LAMPs. HSV-1 DNA was detected in one LAMP negative sample by real-time PCR. One HSV-1 LAMP positive sample was negative in real-time PCR. If real-time PCR was used as a standard, sensitivity of HSV-1, HSV-2, and VZV LAMPs based on microfluidic chip were 90%, 100%, and 100%, and the specificity of three methods were 96%, 100% and 100%, respectively. Conclusion Multiplex LAMP, based on a microfluidic chip that detects three alpha herpesviruses, may be useful in laboratory diagnosis of patients with vesicular skin lesions. Disclosures All Authors: No reported disclosures.

Application of a low-cost, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Plasmodium falciparum imported from Africa

BackgroundChinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa. MethodsWe developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection. ResultsThe new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5–100%] and 99.1% (95% CI 96.7–99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 102 copies/μL, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/μL of blood (equal to 0.04 parasites/μL of DNA) using cultured P. falciparum and 1–7 parasites/μL of blood (4–28 parasites/μL of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays. ConclusionThe novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and cost-effective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

Rapid and simultaneous detection of multiple pathogens in the lower reproductive tract during pregnancy based on loop-mediated isothermal amplification-microfluidic chip

BackgroundFemale reproductive tract infection (RTI) is the common source of varied diseases, especially as an important risk factor for pregnancy outcomes, therefore the rapid, accurate and simultaneous detection of multiple pathogens is in urgent need for assisting the diagnosis and treatment of RTI in pregnant women. Streptococcus agalactiae (S. agalactiae), Enterococcus faecalis (E. faecalis), Gardnerella vaginalis (G. vaginalis), Candida albicans (C. albicans) and Chlamydia trachomatis (C. trachomatis) are five main pathogens in lower genital tract with high risk, serious consequences and clinical demands. The combination of loop-mediated isothermal amplification (LAMP) and microfluidic technology was used to develop the LAMP-microfluidic chip for rapid, simple, sensitive and simultaneous detection of the five target pathogens above.ResultsStandard strains and clinical isolates were used for the establishment of the novel LAMP method in tube and LAMP-microfluidic chip, followed by the chip detection on 103 clinical samples and PCR verification partially. The sensitivities of LAMP of S. agalactiae, E. faecalis, G. vaginalis, and C. albicans in tube were 22.0, 76.0, 13.2, 1.11 CFU/μL, respectively, and C. trachomatis was 41.3 copies/μL; on LAMP-microfluidic chip they were 260, 154, 3.9 and 7.53 CFU/μL, respectively, and C. trachomatis was 120 copies/μL. The positive coincidence rates of clinical stains in tube and on chip experiments were 100%. Compared with the classic culture method performed in hospitals, the positive coincidence rate of the 103 clinical samples detected by LAMP-microfluidic chip were 100%. For the six inconsistent ones, including four G. vaginalis and two C. albicans positive samples tested by LAMP-microfluidic chip and verified by PCR were negative by culturing method in hospitals, indicating the lack of efficient detection by the classic culturing method.ConclusionOur study suggested that the LAMP-microfluidic chips could simultaneously, efficiently, and accurately detect multiple main pathogens, including S. agalactiae, E. faecalis, G. vaginalis, C. albicans and C. trachomatis, in clinical samples of female RTI to give a great clinical value. Accordingly, this novel method has the potential to provide a valuable reference for female RTI screening and early diagnosis during pregnancy.

Development of Colorimetric Loop-Mediated Isothermal Amplification (LAMP) Technique for Rapid, Sensitive and Convenient Detection of Chrysanthemum Chlorotic Mottle Viroid (CChMVd) in Chrysanthemum

Chrysanthemum chlorotic mottle viroid (CChMVd) is a serious pathogen infecting chrysanthemum worldwide. To improve and enhance the detection procedure, the colorimetric loop-mediated isothermal amplifi cation (LAMP) technique was developed. Six LAMP primers were newly designed and tested the optimal conditions using a recombinant plasmid of CChMVd as a DNA template. The optimal conditions for colorimetric LAMP were incubation at 65°C for 60 min. At these conditions, the ladder-liked pattern LAMP products were detected along with the change of color from pink to yellow in the positive reactions. The sensitivity of colorimetric LAMP was up to 2.18 × 103 copies of recombinant plasmid concentration which was 104-fold greater than that of polymerase chain reaction (PCR). The developed colorimetric LAMP was not cross reacted to other viruse s and viroid. Effi ciency of colorimetric reverse transcription (RT)-LAMP for detecting CChMVd in chrysanthemum plantlets obtained from meristem tip culture was evaluated to verify and ensure CChMVd-free plantlets production. The results showed that 18 chrysanthemum plantlets were free of CChMVd contamination. Therefore, the colorimetric RT-LAMP can be used to detect CChMVd routinely due to its accuracy, rapidness and sensitivity.

Development of a loop-mediated isothermal amplification method for detection of Shewanella algae in fish

Shewanella algae is an opportunistic pathogen, which is widely distributed in freshwater and marine environments. Diseases caused by S. algae have resulted in substantial economic losses in aquaculture industry. This study aimed to establish a loop mediated isothermal amplification (LAMP) method for rapid and specific detection of S. algae in aquatic animals. The specific LAMP primers were designed according to the 16 S rRNA gene sequences of S. algae strains deposited in GenBank. Based on adjustment of the primer concentration and optimization of reaction conditions, the LAMP method for S. algae detection was established, and specificity and sensitivity tests were carried out. The optimal temperature of this method was 65 °C at constant 1 h. The LAMP amplification reaction was positive for S. algae, but negative for Pseudoalteromonas pisicida, Aeromonas hydrophila, A. veronii, Vibrio parahemolyticus, S. decolorationis, S. xiamenensis, Enterobacter cloacae, Bacillus cereus and Staphylococcus epidermidis. The minimum concentration of template DNA of S. algae for the LAMP method was 8.15 × 10−5 µg/µL. The LAMP method was comparable to the common PCR when they were used for detection of S. algae in koi carp artificially infected with this pathogen. In this study, a rapid, specific and sensitive LAMP method was successfully established for the detection of S. algae, providing great convenience for rapid diagnosis of aquatic animal diseases caused by S. algae infection.

Diagnostic Performance of Loop-Mediated Isothermal Amplification and Ultrasensitive Rapid Diagnostic Tests for Malaria Screening Among Pregnant Women in Kenya.

Screen-and-treat strategies with sensitive diagnostic tests may reduce malaria-associated adverse pregnancy outcomes. We conducted a diagnostic accuracy study to evaluate new point-of-care tests to screen pregnant women for malaria at their first antenatal visit in western Kenya. Consecutively women were tested for Plasmodium infection by expert microscopy, conventional rapid diagnostic test (cRDT), ultra sensitive RDT (usRDT), and loop-mediated isothermal amplification (LAMP). Photoinduced electron-transfer polymerase chain reaction (PET-PCR) served as the reference standard. Diagnostic performance was calculated and modelled at low parasite densities. Between May and September 2018, 172 of 482 screened participants (35.7%) were PET-PCR positive. Relative to PET-PCR, expert microscopy was least sensitive (40.1%; 95% confidence interval [CI], 32.7%-47.9%), followed by cRDT (49.4%; 95% CI, 41.7%-57.1), usRDT (54.7%; 95% CI, 46.9%-62.2%), and LAMP (68.6%; 95% CI, 61.1%-75.5%). Test sensitivities were comparable in febrile women (n = 90). Among afebrile women (n = 392), the geometric-mean parasite density was 29 parasites/µL and LAMP (sensitivity = 61.9%) and usRDT (43.2%) detected 1.74 (95% CI, 1.31-2.30) and 1.21 (95% CI, 88-2.21) more infections than cRDT (35.6%). Per our model, tests performed similarly at densities >200 parasites/µL. At 50 parasites/µL, the sensitivities were 45%, 56%, 62%, and 74% with expert microscopy, cRDT, usRDT, and LAMP, respectively. This first-generation usRDT provided moderate improvement in detecting low-density infections in afebrile pregnant women compared to cRDTs.

In-Field LAMP Detection of Flavescence Dorée Phytoplasma in Crude Extracts of the Scaphoideus titanus Vector

One of the most destructive diseases affecting grapevine in Europe is caused by Flavescence Dorée phytoplasma (FDp), which belongs to the 16Sr-V group and is a European Union quarantine pathogen. Although many molecular techniques such as loop-mediated isothermal amplification (LAMP) are widely used for the rapid detection of FDp in infected grapevine plants, there is no developed isothermal amplification assay for FDp detection in the insect vectors that are fundamental for the spread of the disease. For this reason, a simple in-field real-time LAMP protocol was optimized and developed for the specific detection of FDp in the insect vector Scaphoideus titanus. The LAMP assay was optimized to work with crude insect extracts obtained by manually shaking a single insect in a buffer for 5 min. Such a simple, sensitive, specific, economic, and user-friendly LAMP assay allowed the detection of FDp in S. titanus in less than half an hour, directly in the field. The developed insect tissue preparation procedure, combined with the LAMP protocol, promptly revealed the presence of FDp in infected S. titanus directly in the vineyards, allowing for monitoring of the spread of the pathogen in the field and to apply timely strategies required for the mandatory control of this pathogen.

Comparison between Polymerase Chain Reaction and Loop Mediated Isothermal Amplification for the detection of Staphylococcus aureus in food

This research aimed to compare Polymerase Chain Reaction (PCR) and Loop Mediated Isothermal Amplification (LAMP) for the detection of Staphylococcus aureus in food. Staphylococcus aureus is a human pathogen due to widespread infection and being methicillin-resistant causing bacteraemia and tissue infections. According to six distinct sequences of the mecA and femA in S. aureus, four specific primers that were designed to detect S. aureus using LAMP. In addition, the sensitivity of LAMP was performed and compared with PCR. The results showed that the LAMP completed the reaction within 30 mins at 65°C. LAMP is considered a promising alternative method compared to the PCR technique where LAMP can be used in laboratories and fields with limited resources.

An endpoint visualization loop-mediated isothermal amplification (LAMP) for detecting bubaline theileriosis

BackgroundTropical theileriosis is a significant disease affecting the health and production levels of buffaloes in India. It is caused by an apicomplexan—Theileria annulata. The timely and accurate detection of infection is vital for implementing a mass vaccination or control programme in a given area under outbreak. Most of the literature concerned with diagnosis of theileriosis revolves around cattle, and practically, there are very limited assays available for detecting bubaline theileriosis. Loop-mediated isothermal amplification (LAMP) assay certainly amplifies the targeted deoxyribosenucleic acid (DNA) with a comparatively higher efficacy, rapidity and sensitivity. Alongside, minimal use of sophisticated instruments in performing LAMP assay is certainly an add on. The present study describes the application of LAMP assay in diagnosing tropical theileriosis in buffaloes alongside, its comparison with polymerase chain reaction (PCR) and blood microscopical examination.ResultsNo cross-reaction was seen with DNA of other haemoprotozoan. LAMP was compared with blood microscopy and PCR. LAMP detected infection in 27 out of 100 buffaloes, while blood microscopy and PCR detected disease in 16 and 24 buffaloes, respectively.ConclusionThe sensitivity, specificity and kappa value prediction of LAMP were found to be much higher than the PCR and blood microscopy. The present communication reports the first use of LAMP in detecting theileriosis in buffaloes in the world.

Development of loop mediated isothermal amplification (LAMP): A new tool for rapid diagnosis of cotton leaf curl viral disease

Cotton leaf curl disease (CLCuD) ranks top among all endemic diseases transmitted by whitefly (Bemisia tabaci) affecting cotton (Gossypium hirsutum) causing severe economic losses to the cotton growers in the Indian subcontinent. For its effective management, robust tools for detection are a prerequisite and it is important to diagnose the virus titre in early stage of infection in plants as well as in the disease transmitting vector. Considering the limitations in current PCR-based techniques we have standardised rapid and sensitive Loop Mediated Isothermal Amplification (LAMP) protocol for the diagnosis of cotton leaf curl virus (CLCuV) in cotton leaves and in its transmitting vector whitefly. Perhaps, this is the first report of use of LAMP tool for rapid diagnosis of CLCuV in cotton and its transmitting vector the whitefly. Further, the colorimetric detection for diagnostic simplicity of amplified LAMP product by using different dyes lead to enhanced applicability of this technique in the field of disease diagnostics. The merit of present study is that the diagnostic failure of PCR and LAMP due to low virus titre in the infected leaf has been circumvented through the combination of rolling circle amplification (RCA) with LAMP. Thus RCA-LAMP can be an option for ultra-sensitive detection of samples with low virus titre. The potential applications of this advanced diagnostic tool in laboratory research on diagnosis of CLCuV, an important viral pathogen of cotton have been discussed.

Development of a Highly Sensitive Loop-Mediated Isothermal Amplification Incorporated with Flocculation of Carbon Particles for Rapid On-Site Diagnosis of Blood Disease Bacterium Banana

Bananas are one of the most crucial fruit crops worldwide and significantly contribute to food security in developing countries. However, blood disease of bananas caused by Ralstonia syzygii subspecies celebensensis has become a threat to banana production. Rapid and accurate diagnosis of BDB for on-site detection is pivotal at an early stage for an effective disease control strategy. This study developed LAMP with specific primers targeting BDB, followed by a flocculation assay for visualising positive amplification in the LAMP assay. The assay was sensitive to picogram amounts of gDNA (0.5 pg). LAMP assay on BDB gDNA showed flocculation, but negative results on Fusarium oxysporus cubense and Ralstonia solanacearum confirming the specificity of the assays. Field testing conducted at MARDI headquarters and Taman Pertanian Universiti discovered that the LAMP-flocculation assays were successful in detecting BDB on symptomatic samples as well as on samples from a healthy plot with no symptom observed at the sampling stage, revealing that this assay can detect BDB at an early infection stage. The validation results showed that the LAMP-flocculation assay was comparable with the PCR technique. This newly developed technique is highly specific and sensitive for the early detection of BDB for the adoption of precautionary control measures.

Improving the specificity of nucleic acid detection with endonuclease-actuated degradation

Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.

Molecular characterization of strawberry vein banding virus from China and the development of loop\u2011mediated isothermal amplification assays for their detection

Strawberry vein banding virus (SVBV) is one of the serious viral pathogens infecting strawberry worldwide. To understand the molecular characterization of SVBV from China, complete genome sequences of sixteen SVBV isolates were cloned and sequenced. Sequence comparison showed they shared high nucleotide sequence identity (93.6–99.5%) with isolates from China and Japan (96.6–98.4%), while relatively low identity with the isolates from Canada (91.9–93.7%) and USA (85.5–85.9%). Phylogenetic analyses based on the complete genome sequence or coat protein (CP) gene showed the SVBV isolates clustered into three clades correlated with geographic distribution. Recombination analyses identified 13 recombinants and 21 recombinant events, indicating frequent and multiple recombinations in SVBV evolution. Furthermore, a sensitive loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of SVBV isolates, which could be especially suitable for seedling propagation, virus-free culture and routine diagnostics in field investigation. This study offers new understanding of the molecular evolution and may help to improve the management of SVBV.

Development of Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick Assay for a Rapid and Sensitive Detection of Cystic Echinococcosis in Livestock in Kenya.

Background Cystic echinococcosis is a zoonotic disease caused by the metacestode stage of Echinococcus granulosus and occurs worldwide, causing considerable economic losses and public health problems. The currently available methods for the diagnosis of animal hydatidosis are time-consuming and require well-equipped laboratories which make them incompatible with testing in resource-poor settings. This study developed and evaluated a rapid, more sensitive, and specific loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the rapid and sensitive detection of cystic echinococcosis. Results In this study, a specific primer set and FITC-labeled probe targeting the conserved region of the NADH-1 gene were designed. The LAMP reaction was performed at 60°C for 40 minutes, and the amplification products were successfully visualized by LFD strips. The analytical sensitivity of LAMP-LFD was determined using 10-fold serial dilutions of E. granulosus DNA. The minimal concentration detected was 10 fg/μl, and no cross-reactivity was observed with DNA extracted from Taenia solium, Taenia saginata, and Fasciola hepatica. The ability of the developed LAMP-LFD assay to detect cystic echinococcosis was further evaluated with 62 cyst samples from slaughtered cattle in Juja Abattoir, Kiambu County, Kenya. The LAMP-LFD was able to detect 59/62 (95.2%, 95% CI 0.87–0.98) as positive samples of E. granulosus compared to 53/62 (85.5%, 95% CI 0.75–0.92) by nested PCR assay. Conclusion Our results indicated that the developed LAMP-LFD technique was more sensitive than the nested PCR assay, rapid, and easy to perform with a simple visual detection of products. Therefore, it could be an important point-of-care diagnostic tool for cystic echinococcosis.