• Journal of Microbiology and Biotechnology
• /
• v.25 no.3
• /
• pp.321-327
• /
• 2015

Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64℃. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10³ CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis.

• Biomedical Science Letters
• /
• v.26 no.3
• /
• pp.149-156
• /
• 2020

We developed a simple and rapid method for detecting vancomycin resistance genes, such as vanA and vanB, using loop-mediated isothermal amplification (LAMP). To identify not only vancomycin resistance genes, but also the genus Enterococcus, primers were designed for vanA, vanB, and 16S rRNA. Screening for vancomycin susceptibility in Enterococcus was performed using Etest (bioMérieux Inc). The results of the LAMP assay were compared to those of real-time RT-PCR. The optimal conditions for the LAMP assay were 65℃ for 60 min. The detection limits of the LAMP assay for vanA, and vanB were 2 × 10² copies/reaction. Compared to RT-PCR, the sensitivities and specificities of LAMP for 16S rRNA, vanA, and vanB were 100/100%, 100/100%, and 100/100%, respectively. The vanA genotype-vanB phenotype accounted for 57.5% (46/80) of the vancomycin-resistant Enterococci samples collected from 2016 to 2019. In conclusion, the LAMP assay developed in this study showed high sensitivity and specificity for vancomycin-resistant genes. Moreover, due to the simplicity and rapidity of the LAMP assay, its use can be very useful in clinical microbiology laboratories.

• Parasites, Hosts and Diseases
• /
• v.51 no.5
• /
• pp.573-577
• /
• 2013

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

• Korean Journal of Agricultural Science
• /
• v.47 no.1
• /
• pp.173-182
• /
• 2020

Human Astrovirus (HuAstV), known as a waterborne virus, is a group IV positive-sense single-stranded RNA that belongs to Astroviridae. The first outbreak of HuAstV was reported in England in 1975. HuAstV can exist not only among clinical patients but also in various water environments, such as water for agriculture and vegetables. For diagnosis of HuAstV from water samples, a polymerase chain reaction (PCR) system has been developed. However, the PCR-based diagnostic method has problems in field application, such as reaction time, sensitivity and specificity. For this reason, in this study we developed the loop-mediated isothermal amplification assay (LAMP) system, aimed specifically at HuAstV. Three prepared LAMP primer sets were tested by specificity, non-specificity and sensitivity; one LAMP primer set was selected with optimum reaction temperature. The developed LAMP primer set reaction conditions were confirmed at 62℃, and detection sensitivity was 1 fg/μL. In addition, restriction enzyme HaeIII (GG/CC) was introduced to confirm that the LAMP reaction was positive. As a result, selected LAMP primer set was 100 - 1000 times more specific, rapid, and sensitive than conventional-nested PCR methods. For verification of the developed LAMP assay, twenty samples of cDNA from groundwater samples were tested. We expect that the developed LAMP assay will be used to diagnose HuAstV from various samples.

• Korean Journal of Veterinary Service
• /
• v.34 no.4
• /
• pp.333-339
• /
• 2011

Loop-mediated isothermal amplification (LAMP) was developed to detect Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacterium (NTM) genomic DNA in blood samples of Korea native cattle. A set of four primers, two outer and two inner, were designed from M. bovis and M. avium genomic DNA targeting the IS6110 and 16S rRNA gene, respectively. Based on 85 Intradermal Tuberculin Test (ITT) positive blood sample and using conventional PCR and LAMP, the agreement quotient (kappa), which measures agreement beyond chance were 0.93 (conventional PCR) and 0.97 (LAMP), respectively. The detection limit of the LAMP method was $2.0{\times}10^2$ copy/ml M. bovis and M. avium cells, compared to $2.0{\times}10^3$ copy/ml M. bovis and M. avium cells for conventional PCR. These results suggest that the LAMP is a powerful tool for rapid, sensitive, and practical detection of MTC and NTM in blood samples of Korea native cattle.

• Research in Plant Disease
• /
• v.28 no.2
• /
• pp.92-97
• /
• 2022

Apple stem grooving virus (ASGV) is a high-risk viral pathogen that infects many types of fruit trees, especially pear and apple, and causes serious economic losses across the globe. Thus, rapid and reliable detection assay is needed to identify ASGV infection and prevent its spread. A reliable reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed, optimize, and evaluated for the coding region of coat protein of ASGV in pear leaf. The developed RT-LAMP facilitated the simple screening of ASGV using visible fluorescence and electrophoresis. The optimized reaction conditions for the RT-LAMP were 63℃ for 50 min, and the results showed high specificity and 100-fold greater sensitivity than the reverse transcription polymerase chain reaction. In addition, the reliability of the RT-LAMP was validated using field-collected pear leaves. Furthermore, the potential application of paper-based RNA isolation, combined with RT-LAMP, was also evaluated for detecting ASGV from field-collected samples. These assays could be widely applied to ASGV detection in field conditions and to virus-free certification programs.

• Genomics & Informatics
• /
• v.20 no.4
• /
• pp.46.1-46.7
• /
• 2022

Influenza A virus (IAV) is the most widespread pathogen causing human respiratory infections. Although polymerase chain reaction (PCR)-based methods are currently the most commonly used tools for IAV detection, PCR is not ideal for point-of-care testing. In this study, we aimed to develop a more rapid and sensitive method than PCR-based tools to detect IAV using loop-mediated isothermal amplification (LAMP) technology. We designed reverse-transcriptional (RT)-LAMP primers targeting the hemagglutinin gene. RNAs from reference H1N1 and H3N2 showed specific RT-LAMP signals with the designed primers. We optimized the reaction conditions and developed universal reaction conditions for both LAMP assays. Under these conditions, the detection limit was 50 copies for both RT-LAMP assays. There was no non-specific signal to 19 non-IAV respiratory viruses, such as influenza B virus, coronaviruses, and respiratory syncytial viruses. Regarding the reaction time, a positive signal was detected within 25 min after starting the reaction. In conclusion, our RT-LAMP assay has high sensitivity and specificity for the detection of the H1 and H3 subtypes, making it suitable for point-of-care IAV testing.

• The Plant Pathology Journal
• /
• v.27 no.4
• /
• pp.385-389
• /
• 2011

A new reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the Potato leafroll virus (PLRV) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to address its advantages over RTPCR. RT-LAMP primers were designed from the open reading frame 3 (ORF3) sequence of PLRV. The RT-LAMP reactions were conducted without or with a set of loop primers. By real-time monitoring using Turbimeter, the RT-LAMP (with loop primers) detects PLRV in less than 30 min, compared to 120 min of RT-PCR. By adding fluorescent reagent during the reaction, final products of the RT-LAMP were fluorescently visualized under UV light or could be differentiated by naked-eye inspection under normal light. The RT-LAMP was extremely sensitive, about 2000-fold more sensitive than RT-PCR. This study presents great potential of the RT-LAMP for diagnosis and PLRV epidemiology because RT-LAMP method is speedy, sensitive, inexpensive, and convenient.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.3
• /
• pp.454-464
• /
• 2019

Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, $MgSO_4$ concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as $89fg/{\mu}l$, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.

• Journal of Life Science
• /
• v.26 no.5
• /
• pp.620-631
• /
• 2016

Viroids are about 250-400 base pair of short single strand RNA fragments have been associated with economically important plant diseases. Due to the lack of protein expression capacity associated with replication, it is very difficult to diagnosis viroid diseases in serological methods. For detecting viroid at plants, molecular-based techniques such as agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), DNA-hybridization, blotting analysis and conventional RT-PCR are reliable. Real-time RT-PCR methods that grafted on RT-PCR methods with improved confirmation methods have been also utilized. However, they are still labor-intensive, time-consuming, and require personnel with expertise. Loop-mediated Isothermal Amplification (LAMP) method is a nucleic acid amplification method under the isothermal condition. The LAMP methodology has been reported to be simple, rapid, sensitive and field applicable in detecting a variety of pathogens. The results of LAMP method can be colorized by adding a visible material such as SYBR green I, Evagreen, Calcein, Berberine and Hydroxy naphthol blue (HNB) with simple equipment or naked eyes. The combination of LAMP method and nucleic pathogens, viroids, can be used to realize simple diagnosis platform for the genetic point-of care testing system. The aim at this review is to summary viroid-caused diseases and the simple visible approach for diagnosing viroids using Loop-mediated Isothermal Amplification (LAMP) method.