Establishment of a Loop-mediated Isothermal Amplification Method for Rice Bacterial Leaf Brown Spot Disease

Rice bacterial leaf brown spot disease caused by Pseudomonas syringae pv.syringae(Pss)is a major disease on rice.In recent years,Pss has emerged worldwide,seriously affecting rice production.It is very important to establish a rapid detection method of Pss for the diagnosis and prevention of this disease.In order to robust and accurately diagnose the rice bacterial leaf brown spot disease in the field and laboratory,an assay system for the Pss was developed in this study,and the specific sequence of hrcN was used as the target,based on loop-mediated isothermal amplification(LAMP).The best detection system was MgSO 48 mmol·L^(-1),Bst DNA polymerase 8 U,dNTP 1.4 mmol·L^(-1),the ratio of internal and outer primers was 2:1,the reaction temperature was 63℃,the reaction time was 45 min,and the lowest sensitivity was 104 CFU·mL^(-1).This results provided an accurate and robust method for laboratory and field diagnosis of bacterial leaf brown spot disease of rice.

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.

Development and Evaluation of a Loop-mediated Isothermal Amplification Assay for the Rapid Detection and Identification of Pectobacterium carotovorum on Celery in the Field

Pectobacterium carotovorum is the causal agent of bacterial soft rot in a wide range of vegetable host species.Once P.carotovorum infects the plant,the spread of the disease is difficult to control.In this study,a rapid and sensitive method based on loop-mediated isothermal amplification(LAMP)was developed for detecting P.carotovorum in celery with soft rot using a primer set designed from the pmrA conserved sequence of P.carotovorum.The specificity of the LAMP primer set for P.carotovorum was extensively validated on both P.carotovorum strains and nontarget strains.The sensitivity was 1 pg of P.carotovorum genomic DNA,which demonstrated 10 times more sensitive than the conventional PCR assay.LAMP was also used to detect P.carotovorum in bacterial suspension.The lowest detection concentration was 104 CFU·mL^−1.In addition,a LAMP assay,in conjunction with a crude DNA extraction method,was successfully performed on P.carotovorum-infected samples derived from both artificially and naturally infected plants.In summary,the LAMP assay established in this study constitutes a simple,sensitive,and rapid method for the detection of P.carotovorum,and has potential application in the control of celery soft rot disease through early detection.

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.

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℃ 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.

Quantum Dot Nanobeads-Labelled Lateral Flow Immunoassay Strip for Rapid and Sensitive Detection of Salmonella Typhimurium Based on Strand Displacement Loop-Mediated Isothermal Amplification

Rapid,sensitive,point-of-care detection of pathogenic bacteria is important for food safety.In this study,we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip(QBs-labelled LFIAS)combined with strand displacement loop-mediated isothermal amplification(SD-LAMP)for quantitative Salmonella Typhimurium(ST)detection.Quantum dot nanobeads(QBs)served as fluorescence reporters,providing good detection efficiency.The customizable strand displacement(SD)probe was used in LAMP to improve the specificity of the method and prevent by-product capture.Detection was based on a sandwich immunoassay.A fluorescence strip reader measured the fluorescence intensity(FI)of the test(T)line and control(C)line.The linear detection range of the strip was 10^(2)–10^(8) colony forming units(CFU)·mL^(-1).The visual limit of detection was 10^(3) CFU·mL^(-1),indicating that the system was ten-fold more sensitive than AuNPs-labelled test strips.ST specificity was analyzed in accordance with agarose gel outputs of polymerase chain reaction(PCR)and SD-LAMP.We detected ST in foods with an acceptable recovery of 85%–110%.The method is rapid,simple,almost equipment-free,and suitable for bacterial detection in foods and for clinical diagnosis.

Reverse Transcription-loop-mediated Isothermal Amplification for Detection of Maize Chlorotic Mottle Virus

Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was established in this study. Based on the sequence of MCMV coat protein coding gene, 3 sets of primers were designed and specificity test showed that the second set of primers was specific to MCMV, Similar sensitivities were observed on RT-LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescence under daylight allows naked easy detection of the amplification of MCMV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCMV in maize seeds, and the result by the addition of calcein was consistent with the result detected by the real time turbidimeter. The method is rapid, specific, sensitive without the need for complicated equipment, and is suitable for rapid field detection of MCMV.

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.

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 （ Chlarnys 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.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

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.

Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay

When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.

Rapid detection of the E198A mutation of carbendazim-resistant isolates in Colletotrichum gloeosporioides by loop-mediated isothermal amplification

Apple bitter rot is a serious agricultural disease caused by Colletotrichum gloeosporioides.In recent years,carbendazim-resistant C.gloeosporioides strains bearing an E198A point mutation in theβ-tubulin gene(GAG to GCG)have emerged,threatening global apple production.As such,rapidly detecting the presence of this E198Amutation in C.gloeosporioides isolates is essential in order to monitor the spread of this pathogen and to prevent outbreaks of disease.Herein,we developed a simple loop-mediated isothermal amplification(LAMP)approach to detecting the E198A mutation in C.gloeosporioides isolates from‘Gala’apple samples.This optimized LAMP protocol was sufficient to establish the E198A genotype of a given isolate following a 60min incubation at 63℃ by using four specific primers.The results of this reaction could be interpreted visually based on a fluorescent yellow-green color change upon the addition of the SYBR Green I dye,and were additionally confirmed via gel electrophoresis.Importantly,this LAMP assay was capable of rapidly and reliably detecting apples that were infected with carbendazim-resistant isolates harboring this E198A mutation.In conclusion,this LAMP assay in this study can rapidly,specifically,and sensitively detect cases of apple bitter rot caused by C.gloeosporioides isolates harboring the E198A mutation.

Preliminary investigation and detection based on loop-mediated isothermal amplification(LAMP)of phytoplasmas associated with diseases in B.napus L.

In the last decade,some disease occurred on our experimental farms that had caused serious losses.They were not caused by fungi,bacteria or viruses.By loop-mediated isothermal amplification(LAMP)technique,the detection results pointed to the possible pathogen as phytoplasma.The investigation results implied that phytoplasmas could cause more than 13 kinds of symptoms in almost all parts of plants in B.napus L.,including witches’broom,multi-stems,aggregate main inflorescences,and flat stems.The incidences of these phytoplasma-associated diseases in our experimental farms rose from 1.61%in 2010 to 6.00%in 2021.Some phytoplasma infected plants died without any growing points.These studies would be helpful for detecting phytoplasmas diseases,selecting disease resistant germplasm and improving varieties with disease resistances in B.napus L.

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.

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.

Establishment of an Improved Loop-mediated Isothermal Amplification Technique for Visual Detection of Brucella

[Objectives]Loop-mediated isothermal amplification(LAMP)was developed and optimized for rapid and specific diagnosis of Brucella infection.[Methods]The conserved sequence of the brucellosis standard strain Omp25 gene fragment was selected,primers needed in the loop-mediated isothermal amplification system were designed,and nucleic acid of the standard strain was extracted.Primers,reaction system,reaction conditions and stain types were repeatedly optimized to establish an improved LAMP detection kit for brucellosis.Confirmatory tests were performed on blood samples from clinically confirmed cases and their sensitivity and specificity were confirmed.Then,the detection technology and kit were applied to the detection and screening of suspected B.melitensis in pastoral areas.Finally,SPSS21.0 was used to evaluate the consistency of LAMP and Q-PCR.[Results]LAMP system was optimized,and the optimal final concentrations of MgSO_(4) and betaine were 8 and 0.8 mmol/L,respectively.The sensitivity and specificity of the modified LAMP technique were verified by using human blood samples with confirmed brucellosis.In LAMP specific test,there was no nonspecific amplification,indicating good specificity.In the LAMP sensitivity test,the minimum detection line for B.melitensis,B.abortus,and B.suis was 10 copies/μL,which was an order of magnitude higher than that for Q-PCR.The consistency between modified LAMP and Q-PCR was 0.941.The established Brucella LAMP kit was used to detect the blood samples of 200 sheep suspected to be infected with Brucella.The LAMP kit detected 42 positive samples,with a positive rate of 21%and a detection sensitivity of 100%.Negative specimens were detected in 158 cases,with a specificity of 100%.There were 41 negative samples detected by PCR,with a positive rate of 20.5%.The detection specificity was consistent with that of LAMP kit.[Conclusions]The modified LAMP for Brucella has been successfully established.This technology is characterized by good sensitivity and specificity,low cost and high efficiency,and is conducive to the development in primary medical institutions.