Loop-mediated Isothermal Amplification Primers Research Articles

Genotyping of 30 kinds of cutaneous human papillomaviruses by a multiplex microfluidic loop-mediated isothermal amplification and visual detection method

BackgroundHuman papillomaviruses (HPVs), a group of non-enveloped small viruses with double-stranded circular DNA which lead to multiple skin diseases such as benign warts, are commonly seen in clinics. The current HPV detection systems aim mainly at mucosal HPVs, however, an efficient clinical approach for cutaneous HPVs detection is lacking.ObjectivesTo establish a rapid detection system for cutaneous HPVs using a colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue (HNB) dye in combination with microfluidic technology.MethodsL1 DNA sequences of the 30 cutaneous HPVs were chemically synthesized, and LAMP primers against L1 DNA were designed with use of an online LAMP designing tool. Isothermal amplification was performed with use of a water bath and the amplification results were inspected with the naked eye. Using PCR sequencing as a control method, the specificity and sensitivity of the new detection system were obtained by detecting clinical samples.ResultsThe lower detection limit of the LAMP assay was 107 viral DNA copies/μl when tested on synthesized L1 DNA sequences, which was better than the conventional PCR. Compared to PCR sequencing, the sensitivity of HPV27, HPV2, HPV1, HPV57, HPV3, HPV4, HPV7 and HPV75 genotypes detections were 100%, whereas the specificity was 34.55, 45.12, 95.83, 98.59 and 97.62% respectively, when tested on clinical samples.ConclusionsThe new cutaneous type HPV detection system is characterized by both a good sensitivity and specificity compared to conventional methods.

Molecular detection of Fusarium graminearum causing head blight of wheat by loop mediated isothermal amplification (LAMP) assay

A rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed for the detection of Fusarium graminearum, predominant Fusarium spp. for causing head blight of wheat in many wheat growing countries including India. The four LAMP primers were designed from partial sequence of Fusarium graminearum chromosome 1, complete genome (accession No. HG970332.2 using region (GenBank Accession No. AY937106.1), unique to F. graminearum. The reaction temperature and ratio of outer/inner primers and optimal Mg2 + concentration for reaction efficiency were standardized. Colorimetric detection of LAMP assay showed the presence of blue colour in F. graminearum, while violet color was observed in negative reaction after adding hydroxyl napthol blue (HNB) dye. Likewise, adding 0.6 mg of ethidium bromide/ml to LAMP products showed full brightness only in positive reactions. Further, a ladder like banding pattern confirms the positive reactions on agarose gel electrophoresis whereas no patterns were observed in negative reactions. Upon amplification in the LAMP assay, only three samples out of eight tested positive by showing blue color and fluorescent signal. The assay has been optimized for speed, sensitivity, and specificity and it can detect less than 100 fg of target DNA template per reaction within 60 min. This could detect latent infection in both wheat inflorescence and seeds. Our results indicated that LAMP offers an excellent method to detect F. graminearum and it can be used as a point of care device for seed quarantine of F. graminearum and providing authentic information for disease management.

Development of a specific isothermal nucleic acid amplification for the rapid and sensitive detection of shrimp allergens in processed food.

Food allergens that cause anaphylactic reactions have become an important health problem worldwide. Among them, shrimp is a popular seafood in many cuisines. The best way to avoid allergic reactions is to mitigate the intake of food allergens. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of shrimp DNA. Using LAMP primers, the identification of shrimp DNA by the LAMP assay was specific and rapid (within 30min). It exhibited no cross-reaction with the DNA of other Crustacea, including crabs and lobster, and at least 0.01% shrimp DNA existed in the test sample. Additionally, the sensitivity of LAMP for detecting shrimp DNA was 100-fold greater than that of conventional PCR. LAMP for the detection of shrimp DNA was reproducible regardless of whether the genomic DNA was extracted from boiled, steamed or roasted shrimp samples. In summary, the LAMP assay established herein not only could be potentially used for diagnosing shrimp DNA but could also be applicable for identifying shrimp allergens in commercial food products in marketplaces.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Background/aim Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methods Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

BackgroundThis study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.ResultsA set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.ConclusionsThe analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Rapid Detection of Cercospora beticola in Sugar Beet and Mutations Associated with Fungicide Resistance Using LAMP or Probe-Based qPCR.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, overreliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance detection "toolbox" for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The LAMP primers specific to C. beticola (Cb-LAMP) assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 day postinoculation. A quinone outside inhibitor (QoI)-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based quantitative PCR (qPCR) assay for detecting an E198A mutation in β-tubulin associated with benzimidazole resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14α-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide-resistant mutants from sensitive wild-type isolates.

The Development of Highly Specific and Sensitive Primers for the Detection of Potentially Allergenic Soybean (Glycine max) Using Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick (LAMP-LFD).

The soybean (Glycine max) has been recognized as a frequent elicitor of food allergy worldwide. A lack of causative immunotherapy of soybean allergy makes soybean avoidance essential. Therefore, sensitive and specific methods for soybean detection are needed to allow for soybean verification in foods. Loop-mediated isothermal amplification (LAMP) represents a rapid and simple DNA-based detection method principally suitable for field-like applications or on-site analytical screening for allergens during the manufacturing of foods. This work describes the systematic development and selection of suitable LAMP primers based on soybean multicopy genes. The chemistry applied allows for a versatile detection of amplified DNA, using either gel electrophoresis, fluorescence recording, or a simple Lateral Flow Dipstick (LFD). LAMP based on the ORF160b gene was highly specific for the soybean and may allow for a detection level equivalent to approximately 10 mg soy per kg food. Various soybean cultivars were detectable at a comparable level of sensitivity. LAMP combined with LFD-like detection facilitates a simple, highly specific and sensitive detection of the soybean without the need for expensive analytical equipment. In contrast to the majority of antibody-based methods for soybean detection, all identified primer sequences and optimized protocols are disclosed and broadly available to the community.

Specific, sensitive and rapid authentication of donkey-hide gelatine (Colla corii asini) in processed food using an isothermal nucleic acid amplification assay

Donkey-hide gelatine (DHG) is a well-known, animal-derived traditional Chinese medicine material called Colla corii asini (known in Chinese as "E'jiao"). Because DHG is claimed to have properties that are beneficial to health, its consumption has increased, but its production has decreased. Thus, the incidence of DHG adulteration has become increasingly serious. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the authentication of DHG. Identification of donkey DNA from DHG was performed specifically and rapidly within one hour by LAMP primers. Moreover, the sensitivity of LAMP in authenticating DHG was 10-3ng, which revealed a 105-fold higher sensitivity than that of conventional PCR. The relative detection limit was 0.1% DHG in the adulterants, including gelatines of horse, cow, pork, goat, sheep or chicken origins. When genomic DNAs extracted from heat-treated DHG samples, including boiling or autoclaving for 40min, were used as templates, DHG detection by LAMP was unchanged and reproducible. In conclusion, the LAMP assay established herein could potentially be applied for the authentication of DHG and DHG-related products in herbal or food markets.

Rapid detection of CITES-listed shark fin species by loop-mediated isothermal amplification assay with potential for field use

Shark fin is a delicacy in many Asian countries. Overexploitation of sharks for shark fin trading has led to a drastic reduction in shark population. To monitor international trade of shark fin products and protect the endangered species from further population decline, we present rapid, user-friendly and sensitive diagnostic loop-mediated isothermal amplification (LAMP) and effective polymerase chain reaction (PCR) assays for all twelve CITES-listed shark species. Species-specific LAMP and PCR primers were designed based on cytochrome oxidase I (COI) and NADH2 regions. Our LAMP and PCR assays have been tested on 291 samples from 93 shark and related species. Target shark species could be differentiated from non-target species within three hours from DNA extraction to LAMP assay. The LAMP assay reported here is a simple and robust solution for on-site detection of CITES-listed shark species with shark fin products.

Detection of Vibrio Pathogens from Cultured Turbot (Scophthalmus maximus) by Loop-Mediated Isothermal Amplification (LAMP)

Abstract Vibrio species are the major concern to the aquaculture industry because they are both fish pathogens and also bring foodborne diseases to human. Loop-mediated isothermal amplification (LAMP) is a simple and rapid method to detect the pathogens. In this study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Vibrio scophthalmi, a pathogen of turbot. 16s rDNA of Vibrio scophthalmi was used as the template to design a set of specific LAMP primers. The LAMP primers were designed, according to our requirements, by Beijing Lanpu Bio-tech Co. Ltd. using Primer Explorer V4 software (http://primerexplorer.jp/e/v4_manual/index.html). The DNA of intestinal pathogens of turbot was extracted using proteinase K kit (Beyotime). By initial trial at 64 ℃ of water bath the reaction become visible in turbid or in fluorescent color after 20 min. Three different turbot fish were positive, indicating that they were containing Vibrio scophthalmi in their intestine and this signifies that all the primers bind specifically to the gene of 16S rRNA gene, in another word they are pathogen positive. To confirm the specific amplification of the 16S rRNA gene, gel electrophoresis was run. The resulted bands were specific to the region of 16SrRNA gene supposed to be amplified. The lowest detection limit of the LAMP assay was the DNA concentration of 1.25 × 10-17 μg/ml. This LAMP method can be of high value to the food industry due to its various advantages such as speed, specificity, sensitivity, cost- and labor-efficiency.

On-Site Detection of Tissues of Buffalo Origin by Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting Mitochondrial Gene Sequences

Meat fraud and adulteration is a growing concern in the marketplace today. Species identification in food product is among primary duties of food office control. In this study, a one-step, species-specific, sensitive, and rapid loop-mediated isothermal amplification (LAMP) assay was developed for identification of tissues of buffalo origin. A set of six LAMP primers were designed by targeting mitochondrial cyt b gene. The reaction temperature of LAMP and component of reaction mixture were optimized as 64 °C amplification temperature, 6 U/μL concentration of Bst DNA polymerase, 3 mM concentration of MgSO4, 0.4 mM concentration of dNTP, 0.6 M concentration of betaine, and 1:6 ratio of outer and inner primer pair. Successful amplifications were confirmed using SYBR Green I dye and agarose gel electrophoresis. The specificity of assay was tested with DNA of cattle, goat, sheep, and pig where amplification was observed exclusively in buffalo. The analytical sensitivity of the LAMP assay for buffalo DNA detection was 0.1 pg. The applicability check of developed assay carried out on known/coded samples and binary meat admixture substantiated the accuracy of assay. Supernatant of tissue exposed to Phire Animal Tissue Direct PCR master mix treatment was observed rich enough with DNA of target species and successfully amplified under optimized LAMP reaction. The comparison of results on the samples undergone pre-standardized species-specific PCR taken as gold standard shown complete match with developed species-specific LAMP assay. Hence, the developed species-specific LAMP assays are effective in detection of tissue of buffalo origin.

Rapid and specific detection of mango (Mangifera indica) in processed food using an isothermal nucleic acid amplification assay

Mango (Mangifera indica) is one of the most popular tropical fruits around the world. It is also widely and commonly used in many cuisines and products in the food industry. However, the anaphylactic reaction caused by mango has long been reported as a major problem for consumption in recent years. To prevent allergens in mango, the best way is to avoid mango in the diet. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of mango in food. Four specifically designed LAMP primers targeting the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions were used to address the LAMP reaction for amplifying mango DNA. The results demonstrated that the detection of mango DNA was specifically validated by the LAMP primer. The sensitivity of LAMP for detecting mango DNA is equivalent to that of the traditional PCR method. The LAMP primer sets showed high specificity for detecting the DNA of mango and had no cross-reactions to other species. Moreover, when mango was mixed with other fruits at different ratios, no cross-reactivity for the detection of mango DNA was manifested during LAMP. Finally, genomic DNAs extracted from different heat-processed mangos were used as templates; the detection of mango DNA by LAMP was not significantly affected and was reproducible. As to this established LAMP herein, mango ingredients can be detected, and commercial foods containing mango can also be identified. This assay will be useful and have potential for the rapid detection of mango DNA in practical food markets.

Simple and visible detection of duck hepatitis B virus in ducks and geese using loop-mediated isothermal amplification

In this study, loop-mediated isothermal amplification (LAMP) was used to establish a rapid, specific, and visual detection method for duck hepatitis B virus (DHBV). The design and synthesis of 4 specific LAMP primers were based on the conserved gene region of the DHBV genome, and the optimum temperature and time of the LAMP reaction were 63°C and 50 min, respectively. The LAMP assay was confirmed to be specific for DHBV detection and had the same sensitivity as the quantitative PCR assay. A visual detection method for rapid determination of results was developed using a color indicator containing phenol red and cresol red. A color change was produced based on a pH change in the reaction system, indicating a positive reaction. For the detection of samples from ducks and geese, the LAMP method has the advantages of simplicity, high sensitivity and specificity, good visibility, and low cost. Moreover, it is more practical and convenient than PCR-related assays for the clinical detection of DHBV.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Pseudomonas syringae pv. tomato in planta

Tomato bacterial speck, caused by Pseudomonas syringae pv. tomato (Pst), is one of the most devastating bacterial diseases in tomato worldwide. To establish a rapid amplification system for the detection of Pst, a loop-mediated isothermal amplification (LAMP) method, which includes two external primers (F3/B3), two internal primers (FIP/BIP) and one backward loop primer (B-Loop), was designed based on the hrpZ gene. The specificity of the LAMP primer set was widely validated on Pst and non-target strains. The conditions for LAMP detection of Pst were optimized to complete in 60 min at 63 °C. The amplification was confirmed through gel electrophoresis or visual inspection using calcein. In the sensitivity test, the detection limit of the LAMP assay was 1.61 × 10 fg μL−1 for genomic DNA and 1.05 × 103 CFU mL−1 for bacterial suspension without DNA extraction. The novel method was also applied for the detection of Pst in artificially and naturally infected tomato leaf and stem tissue samples, and even the early onset of disease could be detected by the assay. This method can rapidly detect Pst-infected tissues without strain enrichment and complex DNA extraction, and hence, it is suitable for quarantine and field detection.

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis.

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

GLAPD: Whole Genome Based LAMP Primer Design for a Set of Target Genomes.

Loop-mediated isothermal amplification (LAMP) technology has been applied in a wide range of fields such as detection of foodborne bacteria and clinical pathogens due to its simplicity and efficiency. However, existing LAMP primer designing systems require a conserved gene or a short genome region as input, and they can’t design group-specific primers. With the growing number of whole genomes available, it is possible to design better primers to target a set of genomes with high specificity based on whole genomes. We present here a whole Genome based LAMP primer designer (GLAPD), a new system to design LAMP primer for a set of target genomes using whole genomes. Candidate single primer regions are identified genome wide and then combined into LAMP primer sets. For a given set of target genomes, only primer sets amplifying them and only these genomes will be output. In order to accelerate the primer designing, a GPU version is provided as well. The effectiveness of primers designed by GLAPD has been assessed for a wide range of foodborne bacteria. GLAPD can be accessed at http://cgm.sjtu.edu.cn/GLAPD/ or https://github.com/jiqingxiaoxi/GLAPD.git. A simple online version is also supplied to help users to learn and test GLAPD: http://cgm.sjtu.edu.cn/GLAPD/online/.

Application of loop-mediated isothermal amplification combined with lateral flow dipstick to rapid and sensitive detection of Alexandrium catenella.

Alexandrium catenella is one of the globally distributed toxic marine microalgae to cause paralytic shellfish poisoning that poses a great threat to marine fisheries, economy, and public health. Development of efficient and sensitive methods for accurate identification of A. catenella to minimize its damage is therefore necessary. In this study, a novel method referred to as loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) (LAMP-LFD) was established for rapid and sensitive detection of A. catenella. The internal transcribed spacer (ITS) gene of A. catenella was cloned for sequencing and used as target for LAMP-LFD. Three sets of LAMP primers (AcLF1, AcLF2, and AcLF3) targeting the ITS were successfully designed, among which AcLF2 displaying the best performance was used in the subsequent tests. A specific LFD probe targeting the amplification region of AcLF2 was further designed. The LAMP-LFD detection system was established and the amplification conditions were optimized. Cross-reactivity tests with common marine microalgae showed that the LAMP-LFD was exclusively specific for A. catenella. The detection limits of LAMP-LFD for A. catenella genomic DNA and the plasmid containing the ITS were 4.63 × 10-4 ng μL-1 and 1.26 × 104 copies μL-1, displaying a sensitivity that is 10 times higher than that of SYBR Green I assay and 100 times higher than that of conventional PCR, respectively. Finally, the practicability of LAMP-LFD was confirmed by test with spiked samples. LAMP-LFD revealed a detection limit of approximately 0.1 cell mL-1, which was 100 times more sensitive than conventional PCR. The optimized LAMP-LFD protocol can be completed within 75 min. Therefore, the established LAMP-LFD is a specific, sensitive, and rapid method that is possibly applicable to the field monitoring of A. catenella.

Loop-Mediated Isothermal Amplification for the Detection of Xanthomonas arboricola pv. pruni in Peaches.

To detect Xanthomonas arboricola pv. pruni, a loop-mediated isothermal amplification (LAMP) detection method were developed. The LAMP assay was designed to test crude plant tissue without pre-extraction, or heating incubation, and without advanced analysis equipment. The LAMP primers were designed by targeting an ABC transporter ATP-binding protein, this primer set was tested using the genomic DNA of Xanthomonas and non-Xanthomonas strains, and a ladder product was generated from the genomic DNA of X. arboricola pv. pruni strain but not from 12 other Xanthomonas species strains and 6 strains of other genera. The LAMP conditions were checked with the healthy leaves of 31 peach varieties, and no reaction was detected using either the peach leaves or the peach DNA as a template. Furthermore, the high diagnostic accuracy of the LAMP method was confirmed with 13 X. arboricola pv. pruni strains isolated from various regions in Korea, with all samples exhibiting a positive reaction in LAMP assays. In particular, the LAMP method successfully detected the pathogen in diseased peach leaves and fruit in the field, and the LAMP conditions were proven to be a reliable diagnostic method for the specific detection and identification of X. arboricola pv. pruni in peach orchards.

Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African swine fever virus (ASFV)

African swine fever (ASF) is a fatal disease caused by a virus in domestic pigs. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay were developed for the detection of African swine fever virus (ASFV). LAMP primers targeting the p10 gene of ASFV were designed, the LAMP reaction system was optimized with plasmid pUC57 containing the p10 gene sequence, and the specificities of the real-time LAMP and the visual assays were tested with the DNA or cDNA of pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV) and ASFV, as well as the plasmid pUC57 containing the p10 gene sequence. The detection limits were determined using a serial dilution of plasmid pUC57 containing the p10 gene sequence. Our results showed that the LAMP assays could accurately and specifically detect ASFV with a detection limit of 30 copies per μl−1 of pUC57 containing p10 gene sequence. In addition, the LAMP assays were further evaluated using various genotypes of ASFV strains. Furthermore, the LAMP assays are comparable with the well-established real-time PCR assay. This study provides promising solutions for facilitating preliminary and cost-effective surveillance for prevention and control of ASFV.

QPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici.

Loose smut of wheat caused by the basidiomycete fungus Ustilago tritici, a seed-borne disease, is difficult to control because of the expanse of wheat planting area and difficulty in pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays are used to rapidly amplify the DNA of U. tritici. Five pairs of primers for qPCR and two series primers for LAMP were designed. Primarily, the specificity of the primer was assessed by using genomic DNA of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Further, the amplification systems were optimized. Finally, the sensitivity of qPCR and LAMP assays were evaluated. The results showed that the primer Y-430 F/R, Y-307 F/R, Y-755 F/R, and Y-139 F/R for qPCR and primers L-139 and L-988 for LAMP could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay was identified as 10 pg μL−1 of genomic DNA, the detection limit for LAMP assay was 100 fg μL−1. We successfully performed qPCR and LAMP assays on wheat loose smut wheat samples. This paper establishes two methods for U. tritici detection, which can be used for diagnosis of wheat loose smut in the laboratory and in the field.