Applications of Loop-Mediated Isothermal DNA Amplification

The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65 degrees C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10(-5) dilution level (equivalent to 17.2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak.

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer.

Human tuberculosis caused by Mycobacterium tuberculosis and Mycobacterium bovis shows similar clinical symptoms; however, the treatment differs because M. bovis is inherently resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. Most available molecular tools cannot distinguish the two biovars. This study addresses this gap by introducing a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography that can simultaneously and differentially detect M. bovis and M. tuberculosis within 60 min. The LAMP method does not require sophisticated high-cost equipment and can be easily implemented in resource-limited settings. Our LAMP facilitates rapid and accurate tuberculosis diagnosis, enabling appropriate therapeutic agents to be selected in areas where bovine and human tuberculosis coexist. It can also screen for M. bovis infection in humans and livestock, providing prevalence data in areas where such information is lacking.

Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the “five forbidden vegetables” (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future.

Rapid Screening for Salmonella in Raw Pet Food by Loop-Mediated Isothermal Amplification

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.

Edwarsiela ictaluri is a potential bacteria as a pathogen causing Enteric Septicemia of Catfish (ESC) and also still a big problem in catfish because it can cause death to 100%. This study aims to develop diagnosis DNA based on molecular using Loop Mediated Isothermal Amplification. Method (LAMP). Detection DNA of E. ictaluri bacteria from 9 isolates was extracted at Fish Health Laboratory, in Depok. used four pieces of specific primers from Eip 18 gene of E. ictaluri. DNA amplification, for LAMP reactions without a thermocycler machine, using only an incubator/water bath/oven, at 65°C for 1 hour. The result of detection with LAMP, showed that 8 isolates were positive of E. ictaluri marked by SYBR green I color change from orange to green. While 1 isolate was negatively marked with SYBR Green remain orange. The LAMP results are also verified with PCR using outer primer F3 and B3 which showed positive E. ictaluri. LAMP can detect ESC disease simple, quickly, easily, economically in 1 hour compared by PCR and LAMP method can be used as an alternative diagnostic test for fish diseases, especially in areas with limited laboratory infrastructure.

Preliminary establishment and validation of a loop-mediated isothermal amplification assay for convenient screening of 13 types of high-risk human papillomaviruses in cervical secretions

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

Development of a novel genotype-specific loop-mediated isothermal amplification technique for Hepatitis B virus genotypes B and C genotyping and quantification

Loop-mediated isothermal amplification (LAMP) method is useful for rapidly detecting Edwardsiella ictalurid infection, especially enteric septicemia of catfish (ESC). This study aims to investigate the LAMP method for E. ictaluri detection in ESC. This research was an experimental study using a total of 55 catfish, consisting of 5 negative controls and 50 catfish injected intraperitoneally with 0.1 mL of E. ictaluri at a concentration of 105 CFU/mL. The kidneys of three fishes were randomly selected 6, 12, 18, 24, 30, 36, 42, 48, and 54 hours after infection with E. ictaluri. In addition, samples were also collected on days 3, 5, and 7. Bacterial analysis was determined by conventional biochemistry (genus and species test), whole-genome sequencing of catfish, and LAMP amplification. All types of data obtained in this study were analyzed by descriptive statistics to compare infected and healthy catfish. The catfish were infected with E. ictaluri after 12 hours of infection according to the LAMP amplification procedure which revealed hemorrhages throughout the body, fins, protruding eyes, necrosis, inflammation of the spleen, liver, intestine, pale gills, gut, abdomen swelling, and tissue necrosis in the upper part of the head. It can be concluded that the LAMP method is more effective than the PCR method for detecting infection with E. ictaluri in catfish.

We developed a novel loop-mediated isothermal amplification (LAMP) method to detect Rickettsia spp., including Rickettsia prowazekii and R. typhi. Species-specific LAMP primers were developed for orthologous genes conserved among Rickettsia spp. The selected modified primers could detect all the Rickettsia spp. tested. The LAMP method was successfully used to detect 100 DNA copies of Rickettsia spp. within approximately 60 min at 63℃. Therefore, this method may be an excellent tool for the early diagnosis of rickettsiosis in a laboratory or in the field.

Qu, G., Fu, S., Shen, N., Wang, J., Xiao, Y., Guan, Y., Tang, N., Chen, L., Gao, S. and Shen, Z. 2010. Rapid and sensitive diagnosis of porcine parvovirus by loop-mediated isothermal amplification (LAMP) method. J. Appl. Anim. Res., 37: 113–116. To form a detection model for the porcine parvovirus (PPV), six special primers were designed based on the PPV gene NS1. After optimization only 45 min were needed to amplify PPV DNA by incubation at 63C. The specificity of the LAMP assay was validated by the absence of any cross-reaction with six other genetically related viruses, followed by restriction digestion and was observed to be similar to PCR assay. The LAMP assay demonstrated 100-fold higher sensitivity compared to PCR, with a detection limit of 12fg DNA. Thus the LAMP can be well-applied to laboratories, as a portable device and valuable tool for differential diagnosis of PPV in the countryside.

Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.