Conventional Loop-mediated Isothermal Amplification Research Articles

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

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

Development of a LAMP method with lateral flow DNA chromatography to diagnose toxoplasmosis in immunocompromised patients

BackgroundRapid and accurate diagnosis of toxoplasmosis is critical, particularly for immunocompromised patients. Several molecular methods could have value for toxoplasmosis diagnosis, but often require sophisticated and expensive equipment, and as such are impractical for use in resource-limited countries. Our study aimed to develop a new rapid diagnostic test for toxoplasmosis that can be used in developed countries as well as low- or middle-income countries.MethodsCommon primers for conventional loop-mediated isothermal amplification (LAMP) and the new LAMP DNA chromatography method were designed based on a 529-bp repeat present in Toxoplasma gondii genomic DNA. A total of 91 clinical samples from 44 patients suspected of having toxoplasmosis who were treated at several hospitals across Japan were tested using the new LAMP DNA chromatography method, conventional LAMP, and nested PCR and the sensitivity and specificity of the methods was compared.ResultsThe LAMP DNA chromatography method showed better sensitivity and specificity (68.2% and 100%, respectively) compared with the nested PCR (45.4% and 100%, respectively) and conventional LAMP (63.6% and 100%, respectively) methods for diagnosis of toxoplasmosis in immunocompromised patients. LAMP DNA chromatography also has better sensitivity and specificity (75% and 100%, respectively) than nested PCR (50.0% and 93.5%, respectively) and conventional LAMP (62.5% and 100%, respectively) to diagnose toxoplasma encephalitis using CSF samples.ConclusionWe developed a LAMP DNA chromatography method to detect T. gondii DNA in clinical samples. This method also successfully detected T. gondii DNA in CSF from patients with toxoplasma encephalitis. This newly developed method can be a valuable rapid diagnostic test for toxoplasmosis in a range of settings, including resource-limited areas like those in low- or middle-income countries.

Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow Biosensor for Streptococci Detection.

Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity. This TFO-LAMP technique was applied for the detection of Group B Streptococcus (GBS). The TFO probe is designed to recognize a specific DNA sequence, termed the TFO targeting sequence (TTS), within the amplified product, facilitating detection via fluorescent instrumentation or lateral flow biosensors. A screening method was developed to identify TFO sequences with high affinity to integrate TFO into LAMP, subsequently incorporating a selected TTS into an LAMP primer. In the TFO-LAMP assay, a FAM-labeled TFO is added to target the TTS. This TFO can be captured by an anti-FAM antibody on lateral flow test strips, thus creating a nucleic acid testing biosensor. The efficacy of the TFO-LAMP assay was confirmed through experiments with specimens spiked with varying concentrations of GBS, demonstrating 85% sensitivity at 300 copies and 100% sensitivity at 30,000 copies. In conclusion, this study has successfully developed a TFO-LAMP technology that offers applicability in lateral flow biosensors and potentially other biosensor platforms.

Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.

Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration.

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses.

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

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

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

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

Enhancement of loop mediated isothermal amplification's sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus

The modified loop-mediated isothermal amplification (LAMP), called multiple hybrid, inner primers (MHP)-LAMP, was developed to enhance the efficiency of the existing LAMP-based assay for Vibrio parahaemolyticus detection. The method was built on a conventional LAMP assay by employing 2 newly designed extra sets of primers to increase the initial binding sites of core primers on the V. parahaemolyticus’s rpoD gene from 8 to 12. With this strategy, the assay detection sensitivity was increased by 10 folds, with the detection limit (DL) approaching 100 copies of purified target genomic DNA (gDNA) as analyzed by real-time turbidity measurement and gel electrophoresis. The MHP also accelerated the rate of DNA amplification by 30%, rendering the assay faster. The MHP-LAMP assay did not cross- react with other pathogens, indicating that it was highly specific for V. parahaemolyticus detection. Whilst V. parahaemolyticus was used as a study model herein, our idea of using MHP to maximize assay sensitivity and speed is considered as a universal strategy that can be applied to enhance efficiency of LAMP-based assays for detecting any DNA and RNA of interest.•The strategy of using multiple hybrid, inner primers (MHP) to enhance LAMP assay's efficiency was demonstrated with success.•The MHP enhanced the sensitivity and speed of the existing LAMP assay, designed to detect V. parahaemolyticus, by 10 times and 30%, respectively.•The proposed strategy can be applied to boost up any other LAMP-based assay's diagnostic performance.

Rapid Visual LAMP Method for Detection of Genetically Modified Organisms.

We developed a novel loop-mediated isothermal amplification (LAMP) method using DNA captured on polyacrylamide microparticles (PAMMPs) as templates (PAMMPs@DNA-LAMP) for rapid qualitative detection of genetically modified organisms (GMOs). Here, DNA was extracted by a fast and cost-effective method using PAMMPs. Four LAMP primers were designed for the PAMMPs@DNA-LAMP method to detect the cauliflower mosaic virus 35S (CaMV35S) promotor in GMOs. We thus developed this method for rapid extraction of DNA (5-10 min) and fast amplification of DNA within ∼30 min at a constant temperature of 63 °C. Moreover, the DNA captured by PAMMPs (PAMMPs@DNA) could be effectively detected by both conventional and quantitative PCR (qPCR) and LAMP. The PAMMPs@DNA-LAMP method was validated with high specificity, sensitivity, and performance for practical sample analysis. This assay detected 0.01% target sequences, which had a high specificity like qPCR and better than the conventional PCR (cPCR). Furthermore, PAMMPs@DNA-LAMP was successfully used to extract and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel PAMMPs@DNA-LAMP assay has been developed, which has higher sensitivity and spends less time than the cPCR detection using the conventional DNA extracted process. This method offers a novel approach for rapid detection of GMOs in the field.

Development of extension-mediated self-folding isothermal amplification technology for SARS-CoV-2 diagnosis

The coronavirus disease (COVID-19) pandemic has highlighted the importance of rapid and accurate diagnosis, and loop-mediated isothermal amplification (LAMP) has become a popular method because of its powerful amplification ability using a simple instrument such as a heater or water bath. However, LAMP has limitations such as the complexity of primer design and the difficulty of designing sequence-specific probes, leading to non-specific amplicons and false-positive results.To overcome these limitations, we developed a novel isothermal amplification system called the Extension-mediated self-folding Isothermal amplification Technology (ExIT). ExIT uses a newly designed, self-folding primer (SP) with two key functions. Hairpin structures are formed when the extended strand of the SP hybridizes, exposing the priming site for continuous binding of the new SP. This results in exponential amplification with only two primers, unlike conventional LAMP primer systems. Additionally, an unnatural base was introduced into the SP, which terminated the extension of polymerase and generated a ssDNA amplicon. This makes it easier to design and apply probes, reducing the possibility of false-positive results even if non-specific amplicons are produced.Through this strategy, we confirmed a sensitivity of 90 copies (3.6 copies/μL) and verified the specificity by testing for the presence or absence of non-complementary targets. Therefore, the validation of the ExIT was completed. In conclusion, ExIT will be key to solving the complexity of conventional LAMP design and offers great potential for successfully introducing sequence-specific probes to improve false positives.

Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM).

Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population's health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.

Low-Temperature Loop-Mediated Isothermal Amplification Operating at Physiological Temperature.

Loop-mediated isothermal amplification (LAMP) is one of the most widely used isothermal amplification technologies in molecular diagnostics. However, LAMP operates at a high temperature of 65 °C; thus, operating LAMP at a lower temperature is desirable to maximize its usefulness for on-site diagnosis. In this study, we propose a new version of LAMP, termed low-temperature LAMP, which operates at the physiological temperature of 37 °C. Low-temperature LAMP differs from conventional LAMP operating at 65 °C in terms of the concentrations of MgSO4 and deoxyribonucleoside triphosphates (dNTPs), as well as the lengths of DNA probes, which are crucial for the execution of low-temperature LAMP. Under the optimal conditions, the amplification efficiency of low-temperature LAMP is comparable to that of conventional LAMP. In addition, the ligation reaction at 37 °C, which is necessary to detect actual target nucleic acids, is combined without altering the temperature, enabling the identification of miR-21, a cancer-promoting oncogenic miRNA, with high sensitivity and selectivity. The method described in this paper does not require expensive DNA modifications or special additives and would facilitate the widespread application of LAMP in facility-limited or point-of-care settings, paving the way to improvements in other isothermal-amplification-based techniques.

A microfluidic-chip-based system with loop-mediated isothermal amplification for rapid and parallel detection of Trichomonas vaginalis and human papillomavirus.

Cervical cancer is a significant global health issue primarily caused by high-risk types of human papillomavirus (HPV). Recent studies have reported an association between Trichomonas vaginalis (T. vaginalis) infections and HPV infections, highlighting the importance of simultaneously detecting these pathogens for effective cervical cancer risk management. However, current methods for detecting both T. vaginalis and HPV are limited. In this study, we present a novel approach using a microfluidic-chip-based system with loop-mediated isothermal amplification (LAMP) for the rapid and parallel detection of T. vaginalis, HPV16, HPV18, and HPV52 in a reagent-efficient and user-friendly manner. Compared to conventional LAMP assays in tubes, our system exhibits enhanced sensitivity with values of 2.43 × 101, 3.00 × 102, 3.57 × 101, and 3.60 × 102 copies per reaction for T. vaginalis, HPV16, HPV18, and HPV52, respectively. Additionally, we validated the performance of our chip by testing 47 clinical samples, yielding results consistent with the diagnostic methods used by the hospital. Therefore, our system not only offers a promising solution for concurrent diagnosis of T. vaginalis and HPV infections, particularly in resource-limited areas, due to its cost-effectiveness, ease of use, and rapid and accurate detection performance, but can also contribute to future research on the co-infection of these two pathogens. Moreover, the system possesses the capability to simultaneously detect up to 22 different types of pathogens, making it applicable across a wide range of domains such as diagnostics, food safety, and water monitoring.

A digital microfluidic platform coupled with colorimetric loop-mediated isothermal amplification for on-site visual diagnosis of multiple diseases.

Traditional diagnosis of infectious diseases relies on advanced analyzers in central hospitals, which is not sufficient for rapid control of epidemics, especially in resource-limited areas, raising the importance of the development of diagnostic systems for point-of-care testing (POCT). Here, we developed a simple and cost-effective digital microfluidic (DMF) platform in combination with colorimetric loop-mediated isothermal amplification (LAMP) for simple on-site diagnosis of diseases by the naked eye. The DMF chip contained four parallel units for the simultaneous detection of multiple genes and samples at a time. After amplification, the results were visualized by endpoint detection with concentrated dry neutral red on the chip. The whole process could be completed within 45 min and the on-chip LAMP reaction was shortened to 20 min. The analytical performance of this platform was evaluated by the detection of Enterocytozoon hepatopenaei, infectious hypodermal and hematopoietic necrosis virus, and white spot syndrome virus genes of shrimp. The DMF-LAMP assay showed a detection limit of 101 copies per μl for each target, exhibiting comparable sensitivity to the conventional LAMP assay but was more efficient. The sensitivity was also competitive with that of microfluidic-based LAMP assays using other POCT devices such as centrifugal discs regarding the detection of the same targets. Moreover, the proposed device possessed a simple chip structure and high flexibility to integrate multiplex analysis, which was beneficial for further widespread use in POCT. The practicability of the DMF-LAMP assay was verified by the testing of field shrimp. The result of the DMF-LAMP assay indicated a good agreement with the qPCR method, with Cohen's kappa values ranging from 0.91 to 1.00 depending on different targets. For the first time, an image processing method was established based on RGB analysis under different lighting conditions, and a universally adaptable positive threshold was determined regardless of lighting conditions. Paired with a smartphone, the objective analytical method was facile to implement in the field. Moreover, the DMF-LAMP system is easy to extend for a broad range of bioassays, with the advantages of low-cost, rapid detection, ease of use, good sensitivity, and easy readout.

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.

Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed. The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1-3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526-2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis.

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.

Internal heating method of loop-mediated isothermal amplification for detection of HPV-6 DNA.

Loop-mediated isothermal amplification (LAMP) is a promising diagnostic tool for genetic amplification, which is known for its rapid process, simple operation, high amplification efficiency, and excellent sensitivity. However, most of the existing heating methods are external for completion of molecular amplification with possibility of contamination of specimens. The present research provided an internal heating method for LAMP using magnetic nanoparticles (MNPs), which is called nano-LAMP. Near-infrared light with an excitation wavelength of 808 nm was employed as the heating source; hydroxy naphthol blue (HNB) was used as an indicator to conduct methodological research. We demonstrate that the best temperature was controlled at a working power of 2 W and 4.8 µg/µL concentration of nanoparticles. The lowest limit for the detection of HPV by the nano-LAMP method is 102 copies/mL, which was confirmed by a gel electrophoresis assay. In the feasibility investigation of validated clinical samples, all 10 positive HPV-6 specimens amplified by nano-LAMP were consistent with conventional LAMP methods. Therefore, the nano-LAMP detection method using internal heating of MNPs may bring a new vision to the exploration of thermostatic detection in the future.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05283-9.

PM 7/20 (3) Erwinia amylovora

PM 7/20 (3) <i>Erwinia amylovora</i>

Fluorescent Detection of Methicillin Resistant Staphylococcus aureus by Loop-mediated Isothermal Amplification Assisted with Streptavidin-coated Quantum Dots.

Background:Methicillin Resistance Staphylococcus aureus (MRSA) could be considered as a major concern in medicine can cause nosocomial infection and bacteremia, especially in patients using catheter and household medical devices.Methods:Using molecular diagnostic methods are important for identification of MRSA from the Methicillin Sensitive Staphylococcus aureus (MSSA). Here we described a fluorescent assay using biotin-labelling Loop-mediated isothermal amplification (LAMP) method assisted with streptavidin-coated Quantum Dots (QDs) for detection of MRSA. For comparison, another fluorescent assay using LAMP assisted with Green Viewer (GV; a fluorescent dye) was applied for detection of MRSA. The mecA gene was selected as the target for amplification by LAMP and for biotin-labeling of the LAMP amplicons, biotin-11-dUTP was mixed with the dNTPs (deoxy Nucleotide Phosphates) in LAMP reaction. For determining the clinical performance of the developed assay, 30 blood samples with MRSA positive results were tested with QD-LAMP, the conventional LAMP, GV-LAMP, and Polymerase Chain Reaction (PCR).Results:Obtained results indicated that % sensitivity of QD-LAMP was 86.66% for detection of mecA positive MRSA samples; however, the Limit of Detection (LoD) of QD-LAMP was 1.5×104 Colony Forming Unit (CFU).Conclusion:The results suggested that the QD-LAMP assay was easy to operate and could be used for detection of MRSA in parallel to the blood culture with less sensitivity for detection of bacteremia and pediatric septicemia with low counts of MRSA.