loop-mediated isothermal amplification
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A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines.
- MeSH
- Circovirus klasifikace genetika izolace a purifikace MeSH
- DNA primery genetika MeSH
- infekce viry čeledi Circoviridae prevence a kontrola veterinární virologie MeSH
- nemoci prasat prevence a kontrola virologie MeSH
- prasata MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- virové vakcíny genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2–6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.
- MeSH
- lidé MeSH
- myši inbrední ICR MeSH
- myši MeSH
- protozoální DNA genetika moč MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- Toxoplasma genetika izolace a purifikace MeSH
- toxoplazmóza diagnóza parazitologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
We show proof of concept for gene targets (polA, tprL, and TP_0619) that can be used in loop-mediated isothermal amplification (LAMP) assays to rapidly differentiate infection with any of the three Treponema pallidum subspecies (pallidum (TPA), pertenue (TPE), and endemicum (TEN)) and which are known to infect humans and nonhuman primates (NHPs). Four TPA, six human, and two NHP TPE strains, as well as two human TEN strains were used to establish and validate the LAMP assays. All three LAMP assays were highly specific for the target DNA. Amplification was rapid (5-15 min) and within a range of 10E+6 to 10E+2 of target DNA molecules. Performance in NHP clinical samples was similar to the one seen in human TPE strains. The newly designed LAMP assays provide proof of concept for a diagnostic tool that enhances yaws clinical diagnosis. It is highly specific for the target DNA and does not require expensive laboratory equipment. Test results can potentially be interpreted with the naked eye, which makes it suitable for the use in remote clinical settings.
- MeSH
- bakteriální proteiny genetika MeSH
- DNA bakterií genetika MeSH
- frambézie mikrobiologie MeSH
- fylogeneze MeSH
- lidé MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- techniky typizace bakterií MeSH
- Treponema pallidum klasifikace genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Poultry Flavivirus (PF) was a recently emerged virus with high morbidity rates and mortality rates in China. It is the causative agent of egg drop syndrome at present. Development of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was the most efficient way to prevent and control the PF disease. The assay was performed at 64 °C for 45 min, using six specific primers that recognized eight targets of the PF E gene. The RT-LAMP assay, compared to conventional reverse transcription polymerase chain reaction, has 100-fold-greater sensitivity, with a detection limit of 1 × 10(-3) copies per μL RNA and no cross-reaction with poultry other viruses. The RT-LAMP assay is a valuable tool for detected PF without requiring any sophisticated equipment, and the detection has potential usefulness for clinical diagnosis in the field.
- MeSH
- diagnostické techniky molekulární metody MeSH
- DNA primery genetika MeSH
- drůbež MeSH
- Flavivirus genetika izolace a purifikace MeSH
- infekce viry z rodu Flavivirus diagnóza veterinární virologie MeSH
- nemoci drůbeže diagnóza virologie MeSH
- proteiny virového obalu genetika MeSH
- senzitivita a specificita MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- veterinární lékařství metody MeSH
- virologie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Čína MeSH
Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen's kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.
- MeSH
- bakteriální geny genetika MeSH
- DNA gyráza genetika MeSH
- DNA primery genetika MeSH
- endodeoxyribonukleasy genetika MeSH
- mastitida skotu diagnóza mikrobiologie MeSH
- mléko mikrobiologie MeSH
- Mycoplasma bovis genetika MeSH
- RNA ribozomální 16S genetika MeSH
- skot MeSH
- techniky amplifikace nukleových kyselin normy MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Bacillus anthracis, the causative agent of anthrax is a Gram-positive, non-motile, spore forming bacterium. Its spores can persist in soil and water for years and can also be aerosolized. A rapid, sensitive and specific method to detect B. anthracis is important for clinical management and preventing spread of anthrax. Loop-mediated isothermal amplification (LAMP) assay is a rapid technique that amplifies target DNA in isothermal conditions with high sensitivity and specificity. In this study, a LAMP assay set targeting a chromosomal and two plasmid markers was developed. The individual assays of the LAMP set targeting pXO1 plasmid (lef), pXO2 plasmid (capB), and chromosome (BA5345) sequences could detect 10, 250, and 100 fg of genomic DNA and 10, 100, and 50 copies of the DNA targets harboured in recombinant plasmids, respectively. The lef and capB LAMP assays could detect ≥ 1 × 103 CFU per mL of bacteria in spiked human blood samples, while BA5345 LAMP assay could detect ≥ 1 × 104 CFU of bacteria per mL of spiked blood. The amplification was monitored in real-time by turbidimeter, and visual detection was also accomplished under normal and UV light after adding SYBR Green 1 dye on completion of the reaction. The assay set was found to be highly sensitive and did not cross-react with the closely related Bacillus spp. and other bacterial strains used in the study.
Phloem-limiting phytoplasmas are known to be causal agents of phyllody, which is recognized by the abnormal development of floral structures resulting in serious yield losses in sesame plants. Currently, identification of the various groups of phytoplasmas that cause sesame phyllody (SP) is conducted by nested PCR, RFLP, and multiplex real-time qPCR assays. However, these methods require intensive labor and are costly and time-consuming so can only be undertaken in well-equipped labs. Here, diagnostic loop-mediated isothermal amplification (LAMP)-based assays allowing rapid detection of specific groups of phytoplasmas within 30 min were developed based on detection of the 16S rRNA sequence of phytoplasmas. Universal 16S rRNA phytoplasma primers and seven primer sets of different 16Sr group phytoplasmas (16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrX, 16SrXI) and universal plant cytochrome oxidase (cox) gene primers were used to detect 16S rRNA group phytoplasma sequences and the cox gene in sesame plants. The LAMP assays were carried out using a real-time fluorometer with amplification plots and annealing curves visualized directly. Results demonstrated that the 16SrI and 16SrII group phytoplasmas were causal agents of sesame phyllody in Vietnam. LAMP-based assays for in-field detection of sesame phyllody-causing phytoplasmas revealed advantages and potential applicability in comparison with conventional approaches. To the best of our knowledge, this is the first assessment of multiple phytoplasma infection associated with sesame phyllody disease in Vietnam using LAMP-based assays.
- MeSH
- diagnostické techniky molekulární MeSH
- DNA bakterií MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- nemoci rostlin MeSH
- Phytoplasma * genetika MeSH
- RNA ribozomální 16S genetika MeSH
- Sesamum * MeSH
- techniky amplifikace nukleových kyselin MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Vietnam MeSH
Trueperella (T.) bernardiae is a well-known bacterial pathogen in infections of humans, rarely in animals. In the present study, five T. bernardiae isolates, isolated from five Peking ducks of four different farms, were identified by phenotypic properties, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and genotypically by sequencing the 16S ribosomal RNA (rRNA) gene, the superoxide dismutase A encoding gene sodA, and the glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. In addition, the T. bernardiae isolates could be identified with a newly developed loop-mediated isothermal amplification (LAMP) assay based on the gyrase encoding housekeeping gene gyrA. All these tests clearly identified the T. bernardiae isolates to the species level. However, the detection of the specific gene gyrA with the newly designed LAMP assay appeared with a high sensitivity and specificity, and could help to identify this bacterial species in human and animal infections in future. The importance of the T. bernardiae isolates for the clinical condition of the ducks and for the problems at farm level remains unclear.
- MeSH
- Actinomycetaceae MeSH
- Arcanobacterium * genetika MeSH
- diagnostické techniky molekulární MeSH
- kachny * genetika MeSH
- RNA ribozomální 16S genetika MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- techniky amplifikace nukleových kyselin MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Peking MeSH
Cíle: Cílem této studie bylo ověření RT-LAMP testů za účelem nahrazení RT PCR testů pro diagnostiku SARS-CoV-2. Typ studie: Metodická studie. Název a sídlo pracoviště: Ústav laboratorní medicíny, Oddělení klinické biochemie, Fakultní nemocnice Ostrava. Metody: Studie zahrnovala 1018 vzorků anonymizovaných pacientů. Izolace virové RNA byla provedena za použití izolačního kitu Automated RNA Isolation Kit na pipetovací stanici Bravo firmy Agilent. Vyizolované vzorky RNA byly použity k detekci viru SARS-CoV-2 pomocí metody RT-PCR za použití diagnostické soupravy COVID-19 Multiplex RT-PCR a metody RT-LAMP pomocí diagnostické soupravy AUMED RT-LAMP Assay SARS-CoV- 2. Výsledky: Metodou RT-PCR bylo detekováno 32.1 % pozitivních vzorků a 67.9 % negativních vzorků. Vedle toho metodou RT-LAMP bylo detekováno 27.9 % pozitivních vzorků a 72.1 % negativních vzorků. Ze 327 pozitivních vzorků identifikovaných pomocí RT-PCR bylo metodou RT-LAMP identifikováno pouze 247 vzorků, 80 vzorků bylo falešně negativních. Z toho vyplývá, že rozdíl mezi metodami je 11.6 %. Test RT-LAMP vykazuje 94.5% specificitu a 75.5% senzitivitu. Obě testované metody vykazují velmi dobrou shodu (k = 0,725) Závěr: Testovaná metoda RT-LAMP, na rozdíl od RT-PCR, není za současných podmínek vhodná pro stanovení SARSCoV-2
Objectives: The aim of this study was to verify whether the RT-LAMP assay for SARS-CoV-2 determination can replace the RT-PCR assay. Design: Methodological study. Settings: Institute of Laboratory Medicine, Department of Clinical Biochemistry, University Hospital Ostrava. Material and methods: The study included 1018 samples of anonymized patients. Viral RNA isolation was performed using the Automated RNA Isolation Kit using an Agilent Bravo pipetting station. The isolated RNA samples were used to detect the virus by RT-PCR using the COVID-19 Multiplex RT-PCR Kit and reverse transcription and loop-mediated isothermal amplification (RT LAMP) using the AUMED test RT-LAMP Assay SARS-CoV-2. Results: 32.1 % of positive samples and 67.9 % of negative samples were detected by the RT-PCR method. In addition, 27.9 % of positive samples and 72.1 % of negative samples were detected by RT-LAMP method. Of the 327 positive samples identified by RT PCR, 247 samples were true positive, but 80 samples were false negative. It follows that the discrepancy of both tested methods was found to be 11.6 %. The RT-LAMP assay showed 94.50% specificity and 75.54% sensitivity. There was a substantial agreement between the two testing methods (k = 0.725). Conclusions: The tested RT-LAMP method, unlike RT-PCR, is not suitable for SARS-CoV-2 determination under current conditions.
