Loop-mediated Isothermal Amplification Amplicons Research Articles

A Closed-Tube Detection of Loop-Mediated Isothermal Amplification (LAMP) Products Using a Wax-Sealed Fluorescent Intercalator

LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.

Loop region-specific oligonucleotide probes for loop-mediated isothermal amplification–enzyme-linked immunosorbent assay truly minimize the instrument needed for detection process

Enteric fever represents a significant public health burden in less-developed countries. Therefore, there is a great need for developing an improved diagnostic tool adapted to the demands of poor-resource clinical laboratories in those countries. The current study has developed a reliable loop-mediated isothermal amplification (LAMP)–enzyme-linked immunosorbent assay (ELISA) for diagnosis of enteric fever with a minimal equipment dependency. The LAMP–ELISA assay involves direct incorporation of a labeled nucleotide into amplicons during the amplification of the SPA3440 gene, their hybridization to the unique tagged oligonucleotide probes during the LAMP reaction, and finally detection of labeled LAMP amplicons by immunoassay technology. Because the designed oligonucleotide probes target the single-stranded DNA segment within the LAMP amplicons, the probe hybridization stage is performed simultaneously with the amplification process. This novel probe design strategy allows both the amplification and hybridization stages to be performed simultaneously and isothermally in a water bath. Among the bacteria tested, positive results were observed only with enteric fever causative bacteria. The LAMP–ELISA assay was successfully applied to artificially contaminated blood samples with a detection limit of 10 colony-forming units (CFU)/ml, which was 100 times more sensitive than polymerase chain reaction (PCR) and turbidity assessment-based conventional LAMP methods. The new assay is considered to be an effective method for diagnosis of enteric fever.

Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe

Electrostatic redox probes interaction has been widely rendered for DNA quantification. We have established a proof-of-principle by using the ruthenium hexaamine molecule [Ru(NH(3))(6)](3+). We have applied this method for real-time electrochemical monitoring of a loop mediated isothermal amplification (LAMP) amplicon of target genes of Escherichia coli and Staphylococcus aureus by square wave voltammetry (SWV). Ruthenium hexaamine interaction with free DNAs in solution without being immobilized onto the biochip surface enabled us to discard the time-consuming overnight probe immobilization step in DNA quantification. We have measured the changes in the cathodic current signals using screen printed low-cost biochips both in the presence and the absence of LAMP amplicons of target DNAs in the solution-phase. By using this novel probe, we successfully carried out the real-time isothermal amplification and detection in less than 30 min for S. aureus and E. coli with a sensitivity up to 30 copies μL(-1) and 20 copies μL(-1), respectively. The cathode peak height of the current was related to the extent of amplicon formation and the amount of introduced template genomic DNA. Importantly, since laborious probe immobilization is not necessary at all, and both the in vitro amplification and real-time monitoring are performed in a single polypropylene tube using a single biochip, this novel approach could avoid all potential cross-contamination in the whole procedure.

Detection of Roundup Ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick

LAMP–LFD, loop-mediated isothermal amplification (LAMP) combined with a lateral-flow dipstick (LFD), was developed and evaluated as a new method for the detection of Roundup Ready soybean (RRS). Biotinylated LAMP amplicons were produced by two sets of six designed primers that specifically recognized the endogenous gene (Lec1) and the event-specific 5′ -junction region (G35S) of RRS followed by hybridization with FITC-labeled probes and LFD detection. The following optimized conditions for the LAMP assay were used: deoxynucleotide triphosphate (dNTP) concentrations ranging from 0.6 to 3.2 mM, 6 mM Mg2+, 4 U Bst DNA polymerase and a 1:6 ratio of outer to inner primers. The LAMP–LFD results were generated within 50 min. The detection limit of LAMP–LFD was 2.4 copies of the linearized plasmid pTLH10 and was 20 times more sensitive than conventional PCR. We demonstrated the high specificity of LAMP–LFD by testing processed soybean products, genetically modified (GM) maize and Bt-cotton meal. The novel LAMP–LFD setup presented here is simple, rapid, and has the potential for future use in the detection of GM ingredients in feed and food products.

Multiplex Loop-Mediated Isothermal Amplification Detection by Sequence-Based Barcodes Coupled with Nicking Endonuclease-Mediated Pyrosequencing

The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.

Detection of <i>Haemophilus parasuis</i> isolates from South China by loop-mediated isothermal amplification and isolate characterisation

Haemophilus parasuis is the etiological agent of Glässer's disease, which is characterised by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal amplification (LAMP) test was developed to improve the specificity, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly amplified the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon could be visualised directly in the reaction tubes following the addition of SYBR Green I dye. The detection limit of this LAMP method was 10 CFU/mL, which was 10 times more sensitive than the earlier 16S rRNA polymerase chain reaction (PCR) test conducted by Oliveira, Galina and Pijoan (2001), and no cross-reactivity was observed from other non-H. parasuis strains. This LAMP test was evaluated further on 187 clinical specimens from pigs suspected of being infected with H. parasuis. Forty-three were found positive by bacterial isolation of H. parasuis, as well as by the 16S rRNA PCR and LAMP tests. The 43 H. parasuis isolates were classified into 9 serovars and had 37 genetic patterns when analysed by pulsed-field gel electrophoresis (PFGE). This displayed that various H. parasuis serovars and genotypes were widely distributed in South China. Therefore, the speed, specificity and sensitivity of the LAMP test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of H. parasuis in favour of controlling Glässer's disease.

DNA circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform.

This article describes the use of non-enzymatic nucleic acid circuits based on strand exchange reactions to detect target sequences on a paperfluidic platform. The DNA circuits that were implemented include a non-enzymatic amplifier and transduction to a fluorescent reporter; these yield an order of magnitude improvement in detection of an input nucleic acid signal. To further improve signal amplification and detection, we integrated the enzyme-free amplifier with loop-mediated isothermal amplification (LAMP). By bridging the gap between the low concentrations of LAMP amplicons and the limits of fluorescence detection, the non-enzymatic amplifier allowed us to detect as few as 1200 input templates in a paperfluidic format.

Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpoS gene

A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated for the detection of Vibrio vulnificus. Biotinylated LAMP amplicons were produced by a set of six designed primers that recognized the V. vulnificus RNA polymerase subunit sigma factor S ( rpoS) gene followed by hybridization with an FITC-labeled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP–LFD method accurately identified 14 isolates of V. vulnificus but did not detect 25 non- vulnificus Vibrio isolates and 37 non -Vibrio isolates. The sensitivity of LAMP–LFD for V. vulnificus detection in pure culture was 1.5 × 10 3 CFU ml −1 or equivalent to 2.8 CFU per reaction. In the case of spiked oyster samples without enrichment, the detection limit for V. vulnificus was 1.2 × 10 4 CFU g −1 or equivalent to 11 CFU per reaction. The results show that this method appears to be accurate, precise and valuable tool for identification of V. vulnificus and can be used efficiently for detection of V. vulnificus in contaminated food sample.

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin antibodies. Being homozygous for the haptoglobin gene deletion allele (HP(del)) is the only known cause of congenital anhaptoglobinemia, and detection of HP(del) before transfusion is important to prevent anaphylactic shock. In this study, we developed a loop-mediated isothermal amplification (LAMP)-based screening for HP(del). Optimal primer sets and temperature for LAMP were selected for HP(del) and the 5' region of the HP using genomic DNA as a template. Then, the effects of diluent and boiling on LAMP amplification were examined using whole blood as a template. Blood samples diluted 1:100 with 50 mmol/L NaOH without boiling gave optimal results as well as those diluted 1:2 with water followed by boiling. The results from 100 blood samples were fully concordant with those obtained by real-time PCR methods. Detection of the HP(del) allele by LAMP using alkaline-denatured blood samples is rapid, simple, accurate, and cost effective, and is readily applicable in various clinical settings because this method requires only basic instruments. In addition, the simple preparation of blood samples using NaOH saves time and effort for various genetic tests.

Detection of Group 1 Trypanosoma brucei gambiense by Loop-Mediated Isothermal Amplification

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3' end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ∼1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 10(3) trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test.

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non-parahaemolyticus Vibrio isolates and 35 non-Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml⁻¹. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 x 10³ CFU g⁻¹ or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.

Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes

Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10 2 copies of the target DNA to less than 70 min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.

FRET-Based Assimilating Probe for Sequence-Specific Real-Time Monitoring of Loop-Mediated Isothermal Amplification (LAMP)

Isothermal nucleic acid amplification offers compelling opportunities for simple gene-based diagnostics, although the technologies for monitoring these reactions in real-time are still relatively undeveloped compared to PCR-specific probes. In this article, we describe the adaptation of an innovative FRET-based probe architecture for monitoring the isothermal LAMP reaction in real-time, allowing single-step, direct identification of different isolates of the agricultural pathogen Ralstonia solanacearum. The probe design is analogous to a two-component molecular zipper used for monitoring rolling circle amplification (RCA), in which a quenching oligonucleotide strand is displaced from a fluorescent strand by a strand-displacing polymerase during the reaction. Because the fluorescent strand is stably assimilated into the LAMP amplicon in our configuration, we use the nomenclature assimilating probe to describe the probe. Using these probes, we have demonstrated the ability to perform real-time, quantitative LAMP analysis of DNA, detecting fewer than ten gene copies. Probe compositions were optimized to prevent noticeable inhibition of the LAMP reaction, allowing observation of kinetics for different LAMP reactions and inference of primer design rules for accelerated detection. The sequence-specific nature of the assimilating probes offers compelling advantages over non-specific methods for real-time detection of LAMP amplification, including improved selectivity and the potential for multiplexed detection.

A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification

This study reports a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combing nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic bead-based microfluidic system. By using specific probe-conjugated magnetic beads, the target deoxyribonucleic acid (DNA) of the MRSA can be specifically recognized and hybridized onto the surface of the magnetic beads which are then mixed with clinical sample lysates. This is followed by purifying and concentrating the target DNA from the clinical sample lysates by applying a magnetic field. Nucleic acid amplification of the target genes can then be performed by the use of a loop-mediated isothermal amplification (LAMP) process via the incorporation of a built-in micro temperature control module, followed by analyzing the optical density (OD) of the LAMP amplicons using a spectrophotometer. Significantly, experimental results show that the limit of detection (LOD) for MRSA in the clinical samples is approximately 10 fg μL(-1) by performing this diagnostic assay in the magnetic bead-based microfluidic system. In addition, the entire diagnostic protocol, from bio-sample pre-treatment to optical detection, can be automatically completed within 60 min. Consequently, this miniature diagnostic assay may become a powerful tool for the rapid purification and detection of MRSA and a potential point-of-care platform for detection of other types of infections.

COMPARATIVE STUDY OF SPECIFICITY AND SENSITIVITY FOR IHHNV DETECTION BETWEEN REAL-TIME PCR AND LAMP METHODS

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the most important pathogens infecting penaeid shrimp and causes huge economic losses in the shrimp culture industry worldwide. Because no commercial vaccine is yet available, the most effective way of containing the disease is by the routine screening of juveniles and adults for the presence or absence of the virus. Therefore, our goal is to establish a simple and rapid examination system for infectious hypodermal and hematopoietic necrosis virus in places such as shrimp ponds. Two detection methods, real-time PCR and loop-mediated isothermal amplification (LAMP), were developed for IHHNV diagnosis, and then their specificity and sensitivity were compared in the study. Using the real-time PCR method, the assay had a detection limit of six copies of DNA template of IHHNV, and had a correlation coefficient of 0.99521 between template concentration and threshold cycle value at the template concentration of 6.038×104 to 6.038×109cps/mL. Furthermore, the approach had no signal response to genomic DNA of white spot syndrome virus and shrimp. The result revealed that the detection method had high specificity and sensitivity for IHHNV detection. However, the costly real-time thermal cycler and technically demanding were deemed to be not appropriate for IHHNV detection in field conditions. LAMP was a novel, sensitive and rapid detection technique and could be applied for disease diagnosis in aquaculture. Here, a set of four primers was designed using PrimerExplorer V4 software by targeting the IHHNV genome DNA, and used to develop the LAMP method for IHHNV detection. Using the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64℃. Also, the LAMP amplicon was observed directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. Sensitivity assay showed that the method also had a detection limit of six copies of DNA template of IHHNV. Moreover, genomic DNA of white spot syndrome virus and shrimp were unable to be detected within 60min using the LAMP method. Overall, these data revealed that the LAMP method had an equivalent to the real-time PCR method in specificity and sensitivity for IHHNV detection. Considering that the LAMP method had great advantage in its performance and low cost, this technique was more suitable for IHHNV detection in field conditions. Therefore, the LAMP method could be applied routinely to check the shrimp in hatcheries and growout ponds, so that virus carrying shrimp could be found during the early infection stages and counter measures could be devised before the infection becomes epizootic.

Development and evaluation of a loop‐mediated isothermal amplification assay for rapid detection of chicken anaemia virus

Chicken anaemia virus (CAV) causes an economically important viral disease in chickens worldwide. The main aim of this study was to establish a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for detecting CAV infection. A set of four specific LAMP primers were designed based on the nucleotide sequence of the CAV VP2 gene, which encodes a nonstructural protein. These were used for the amplification of a specific target region of the VP2 gene. LAMP amplicons were successfully amplified and detected by DNA electrophoresis and by direct naked eye SYBR Green I visualization. A sensitivity test systematically demonstrated that the LAMP assay was superior to a conventional PCR assay with a minimum concentration limit of 100 fg compared to 10 ng for the conventional PCR. The specificity of the LAMP assay for CAV detection is consistent with conventional PCR. Using this established LAMP assay, infected and uninfected clinical samples obtained from an experimental farm were fully verified. A novel nucleic acid-based approach of LAMP assay was successfully developed for detecting CAV infection. In this study, these results indicate that the developed LAMP assay herein for CAV detection is a time-effective, simple, sensitive and specific test that can be used as an alternative approach in the future for large-scaled diagnosis on the farm of CAV infection.

Detection of African swine fever virus by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of African swine fever virus (ASFV). This assay targets the topoisomerase II gene of ASFV and its specificity was confirmed by restriction enzyme digestion of the reaction products. The analytical sensitivity of this ASFV LAMP assay was at least 330 genome copies, and the test was able to detect representative isolates of ASFV ( n = 38) without cross-reacting with classical swine fever virus. The performance of the LAMP assay was compared with other laboratory tests used for ASF diagnosis. Using blood and tissue samples collected from pigs experimentally infected with ASFV (Malawi isolate), there was good concordance between the LAMP assay and real-time PCR. In addition to detecting the reaction products using either agarose gels or real-time PCR machines, it was possible to visualise dual-labelled biotin and fluorescein ASFV LAMP amplicons using novel lateral flow devices. This assay and detection format represents the first step towards developing a practical, simple-to-use and inexpensive molecular assay format for ASF diagnosis in the field which is especially relevant to Africa where the disease is endemic in many countries.

Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method

A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10 pg of Acanthamoeba DNA could be detected, 10 times more sensitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2 h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection.

Shrimp hepatopancreatic parvovirus detection by combining loop-mediated isothermal amplification with a lateral flow dipstick

Present methods such as traditional PCR, PCR-ELISA, real-time PCR and histopathology for detection of shrimp hepatopancreatic parvovirus (PmDNV) entail various disadvantages including high cost, long assay time or use of toxic substances. Loop-mediated isothermal amplification (LAMP) of target nucleotide sequences under inexpensive isothermal conditions combined with amplicon detection by chromatographic lateral flow dipsticks (LFD) allowed simpler detection within 75 min. Biotinylated LAMP amplicons from the targeted portion of the PmDNA capsid protein gene were produced under isothermal conditions at 63 °C for 1 h and then hybridized at 63 °C for 5 min with an FITC-labeled probe (optimized at 20 pmol) that was specific for the LAMP amplicons (i.e., outside the primer region). The FITC-labeled, biotinylated LAMP product picked up gold-labeled, anti-FITC near the LFD origin and the whole, triple-labeled complex was captured by an immobilized biotin-binding protein to yield a red nano-gold stripe at the LFD test line. With a DNA template derived from PmDNV-infected shrimp, the LAMP–LFD detection limit was 1 ng while that for one-step PCR-electrophoresis was 10 ng. Comparative sensitivity for one nested-PCR-electrophoresis method was 1 ng but for another 0.1 ng. The LAMP–LFD method gave negative test results with DNA extracts from normal shrimp and from shrimp infected with other DNA viruses including monodon baculovirus (MBV), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish.

BackgroundEnteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel.ResultsA loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish.ConclusionThe ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.