One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay

A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV-containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.

Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification assays for Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 in Clinical Samples

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.

Rapid,sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming.The rapid detection and identification of V.anguillarum,and other pathogens that infect marine organisms,is crucial to effective disease management.In this study,we developed a loop-mediated amplification (LAMP) assay to detect V.anguillarum in an hour in a single tube without the need for thermal cycling.Conserved regions of the metalloproteinase (empA) gene of V.anguillarum served as the targets for primer design.A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase.In the optimized LAMP assay,6.7 pg of V.anguillarum DNA could be detected.Six strains of V.anguillarum and 17 strains of non-V.anguillarum bacteria were used in this study to evaluate the species specificity of the primers.The six V.anguillarum strains gave a positive result in the LAMP assay.This method was also validated in V.anguillarum-infected fish.This LAMP method is more sensitive than PCR in the detection of V.anguillarum and shows good species specificity.The LAMP assay is therefore an effective method for the quick detection of V.anguillarum both in the laboratory and in the field.

Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica

Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.

Detection of Staphylococcus aureus in Dairy Food by Loop-mediated Isothermal Amplification

[ Objective ] This study was to investigate the creditability of applying loop-mediated isothermal amplification method （LAMP） to detect Staphylococcus aureus in dairy products. [ Methods] The primers for heat resistant nuclease gene （nuc） of Staphylococcus aureus were designed for establishing the LAMP method for rapidly detecting Staphylococcus aureus. [ Results ] The results of LAMP detection on Staphylococcus aureus in various dairy products were completely identical with that by bacterial isolation test; meanwhile it has a high specificity and a 10-fold sensitivity over the hemi-nested PCR. [ Conclusion] LAMP can be used for the detection of Staphylococcus aureus in dairy products.

Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop (Chlamys farreri).

Rapid,specific and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification targeted to vvhA gene

Vibrio vulnificus is an estuarine bacterial pathogen for human.The rapid,specific and sensitive detection of V.vulnificus is urgently needed for early disease diagnosis and timely treatment of V.vulnificus infection.In the study,a loop-mediated isothermal amplification（LAMP） technique was developed for V.vulnificus detection with a set of primers,composed of two out primers and two inner primers targeted to vvh A gene.The optimal amplification temperature was 63°C and the reaction only took 35 min.The amplification products could not only be detected by agarose gel electrophoresis with ladder-like pattern bands,but also could be visualized using calcein with naked eye directly.Forty-five strains were tested for the specificity of LAMP assay,and all the V.vulnificus strains were identified correctly while other strains were negative results.The sensitive of the new LAMP assay was 100-fold more sensitive than the conventional PCR.Meanwhile,all the V.vulnificus strains were detected correctly in spiked,clinical and environmental samples by the new LAMP assay.Compared with other well-known techniques,the new LAMP assay targeted to vvh A gene was extremely rapid,simple,sensitive and specific for V.vulnificus identification.

Rapid detection of Shigella and Salmonella in rhesus monkeys by loop-mediated isothermal amplification assay

Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.

Sensitive and rapid detection of two toxic microalgae Alexandrium by loop-mediated isothermal amplification

A loop-mediated isothermal amplification （LAMP） assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning （PSP）. Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer （ITS） of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65℃. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection lim- its of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction （PCR） method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum.

The Development of a Loop-Mediated Isothermal Amplification (LAMP) Procedure for Plague Diagnostic

Plague caused by Yersinia pestis is one of the infectious diseases subject to the International Health Regulations (IHR). Permanent monitoring of the focal plague areas is mandatory in order to enable prompt control measures to prevent the spread of the disease. Therefore, the availability of efficient diagnosis tests is of paramount importance. Here, we describe a loop-mediated isothermal amplification (LAMP)-based procedure for rapid Y. pestis detection. We constructed a set of LAMP primers, which were used in assays to establish the reaction conditions that would lead to the quick visualization of the results by evaluating the test tube with the naked eye. The primers were specifically designed to target the caf1 gene located on pFra/Tox (pMT), a prototypical plasmid of Y. pestis. The LAMP procedure was performed at 65°C for 45 min in a water bath and allowed for the detection of at least 10 pg of bacterial DNA. Due to its simplicity, specificity, sensitivity and rapidity, the LAMP technique is an additional tool that may be implemented in routine plague diagnoses, especially in emergencies.

Efficient detection of pathogen virus in sand dabs,Paralichthys olivaceus using loop-mediated isothermal amplification(LAMP)

Viral hemorrhagic septicemia virus（VHSV） and marine birnavirus（MABV） are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of pathogen infection. The loop-mediated isothermal amplification（LAMP） of DNA is currently one of the most commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal conditions. In the present study, a novel and highly specific LAMP assay for the sensitive and rapid detection of VHSV and MABV infection in fish was developed. Using a set of synthesized primers matching a specific region of the genome, the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficient detection of VHSV and MABV infection in the wild fish, Paralichthys olivaceus. Our results demonstrate the simplicity and convenience of this method for the detection of viral infection in aquatic organisms.

Rapid and Visualized Detection Method of Canine Parvovirus Using Loop-Mediated Isothermal Amplification

[Objective] To develop a rapid and visualized detection method using loop-mediated isothermal amplification （LAMP）, improve the detection rate of canine parvovirus （CPV） and reduce the testing cost. [ Method] According to the conserved regions of the VP2 gene of CPV, six primers were designed to amplify the special DNA sequences by LAMP. In addition, the reaction conditions of LAMP were optimized, and the sensitivity, specificity, repeatability and stability were verified. [ Result] The optimal reaction time of the LAMP method for CPVwas 60 min. The products obtained by LAMP had high specificity without cross-reaction with other generic viruses. The sensitivity of the LAMP was 100 times higher than that of PCR. [ Conclusion] The LAMP method for detecting CPV has high practical value. It has many advantages such as high specificity, high sensitivity, simple operation, low cost and rapid analysis, and it does not require special equipment. Therefore, this method is more suitable for the detection of CPV.

Development of Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid and Visualized Detection of Classical Swine Fever Virus

[ Objective] To develop a rapid and visualized detection method of classical swine fever virus （CSFV） using reverse transcriptase loopmediated isothermal amplification （RT-LAMP）. [ Method ] A total of six special primers were designed based on the conserved sequences of CSFV gene. After optimizing, the reaction of RT-LAMP was carded out at 63℃ for 45 rain. The RT-LAMP products were analyzed by agarose gel electro- phoresis. The sensitivity, specificity and repeatability were verified, respectively. [ Result] The RT-LAMP method could be used for detecting CSFV rather than six generic viruses. The sensitivity of RT-LAMP was 100 times higher than that of RT-PCR. The detection of 27 clinical samples by RT- LAMP and RT-PCR showed that RT-LAMP is more reliable and convenient. [ Conclusion] The RT-LAMP method is sensitive and reliable for the detection of CSFV.

Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Ampliflcation(RT-LAMP) on a Chip from Whole Blood

Viral load measurements are an essential tool for the long-term clinical care of hum an immunodeficiency virus(HIV)-positive individuals. The gol d standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification(RT-LAMP) on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RTLAMP droplet, corresponding to a whole blood concentration of 670 viruses per μL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a flnger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation.

Detection of coat protein gene of nervous necrosis virus using loopmediated isothermal amplification

Objective:To establish a novel and highly specific loop-mediated isothermal amplification(LAMP) assay for the identification of nervous necrosis virus(NNV) infection.Methods:A set of synthesized primers was used to match the sequences of a specific region of the nnr gene from the National Center for Biotechnology Information database,not originating from NNV-infected fish,the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time.In addition,to determine species-specific LAMP primers,cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus.Results:The optimized LAMP reaction carried out at 64 ℃ for 60 min,and above 4 U Bst DNA polymerase.The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction.The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus.Conclusions:The development of LAMP primers based on genetic information from a public database,not virus-infected samples,may provide a very simple and convenient method to identify viral infection in aquatic organisms.

Rapid identification of Dendrobium officinale using Loop-Mediated Isothermal Amplification (LAMP) method

Dendrobium officinale is not only an ornamental plant, but also a valuable medicinal herb that is widely used in traditional Chinese medicine. However, distinguishing D. officinale from other Dendrobium species is usually a difficult task. In this study,we developed a rapid identification protocol for D. officinale using the loop-mediated isothermal amplification(LAMP) method. A set of primers were specifically designed to detect a modified internal transcribed spacer region of D. officinale at 65 ℃ within 40 min after adding SYBR~? Green I, which was used for the detection of D. officinale. Unlike commonly used adulterants, reaction mixtures containing D. officinale DNA changed from orange to green, and this color change was easily observed with the naked eye. Thus, this methodology can be used to accurately differentiate D. officinale from other Dendrobium species, is quick as all D. officinale samples were amplified within 40 min, and specific as samples of the adulterants were not amplified. The specificity of this LAMP-based method was confirmed by testing 17 samples of D. officinale and 32 adulterant samples from other Dendrobium species. This LAMP-based rapid identification method does not require expensive equipment or specialized techniques and can be used in field surveys for accurate and fast on-site identification.

A Pilot Study of Quantitative Loop-mediated Isothermal Amplification-guided Target Therapies for Hospital-acquired Pneumonia

Background： It is important to achieve the definitive pathogen identification in hospital-acquired pneumonia （HAP）, but the traditional culture results always delay the target antibiotic therapy. We assessed the method called quantitative loop-mediated isothermal amplification （qLAMP） as a new implement for steering of the antibiotic decision-making in HAP. Methods： Totally, 76 respiratory tract aspiration samples were prospectively collected from 60 HAP patients. DNA was isolated from these samples. Specific DNA fragments for identifying 11 pneumonia-related bacteria were amplified by qLAMP assay. Culture results of these patients were compared with the qLAMP results. Clinical data and treatment strategies were analyzed to evaluate the effects of qLAMP results on clinical data. McNemar test and Fisher＇s exact test were used for statistical analysis. Results： The detection of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Stenotrophomonas maltophilia, Streptococcus pneumonia, and Acinetobacter baumannii by qLAMP was consistent with sputum culture （P 〉 0.05）. The qLAMP results of 4 samples for Haemophilus influenzae, Legionella pneumophila, or Mvcoplasma pneumonia （MP） were inconsistent with culture results; however, clinical data revealed that the qLAMP results were all reliable except 1 MP positive sample due to the lack of specific species identified in the final diagnosis. The improvement of clinical condition was more significant （P 〈 0.001） in patients with pathogen target-driven therapy based on qLAMP results than those with empirical therapy. Conclusion： qLAMP is a more promising method for detection of pathogens in an early, rapid, sensitive, and specific manner than culture.

Gold Nanoparticles Based Colorimetric Detection of Target DNA After Loop-mediated Isothermal Amplification

We have developed a rapid, simple and label-free colorimetric method for the identification of target DNA It is based on loop-mediated isothermal amplification（LAMP）. Plain gold nanoparticles（AuNPs） are used to indicate the occurrence of LAME The amplified product is mixed with AuNPs in an optimized ratio, at which the deoxyribonucleotides（dNTPs） bind to the AuNPs via ligand-metal interactions and thus enhance AuNPs stability. If a target DNA is amplified, the dramatic reduction of the dNTPs leads to the aggregation of AuNPs and a color change from red to blue. The success of the method strongly depends on the ionic strength of the solution and the initial concentration of dNTPs. Unlike other methods for the identification of isothermal products, this method is simple and can be readily applied on site where instrumentation is inadequate or even lacking.