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

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

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

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

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

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.

• MeSH
• antrax * mikrobiologie   prevence a kontrola   MeSH
• Bacillus anthracis * genetika   MeSH
• diagnostické techniky molekulární * normy   MeSH
• DNA bakterií genetika   MeSH
• senzitivita a specificita MeSH
• techniky amplifikace nukleových kyselin * normy   MeSH
• Publikační typ
• časopisecké články 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

Úvod: Cílem práce je porovnání senzitivity detekce mikrometastáz v hilových a mediastinálních lymfatických uzlinách primárních (nemalobuněčných) a sekundárních (metastázy kolorektálního karcinomu) nádorů plic pomocí standardního histopatologického vyšetření v barvení hematoxylin- eosin, imunohistochemického vyšetření s protilátkou proti cytokeratinu 19 a metodou One-Step Nucleic Acid Amplification. Metoda: Při radikální chirurgické léčbě nemalobuněčných primárních plicních karcinomů a metastáz kolorektálního karcinomu byly u 100 pacientů zařazených do studie v období 2015–2017 podle standardního schématu odebrány hilové a mediastinální lymfatické uzliny. Tyto uzliny pak byly v podélné ose děleny na 4 stejné části, přičemž část první a třetí zleva byla určena k vyšetření metodou One-Step Nucleic Acid Amplifi cation, část druhá a čtvrtá k histologickému vyšetření. Při histologickém vyšetření byly příslušné části uzlin nejprve vyšetřeny standardním postupem v barvení hematoxylin-eosin a následně pak i imunohistochemicky s protilátkou proti cytokeratinu 19. Metoda One-Step Nucleic Acid Amplification byla prováděna soupravou firmy Sysmex (Kobe, Japonsko), jejím principem je detekce mRNA (messenger ribonucleic acid) cytokeratinu 19 reverzní transkripcí spojenou s izotermickou amplifikací. Výsledky: Celkem bylo nemocným zařazeným do studie odebráno a uvedenou metodikou vyšetřeno 1426 lymfatických uzlin. Shodné výsledky vyšetření hematoxylinem-eosinem, imunohistochemií s protilátkou proti cytokeratinu 19 a One-Step Nucleic Acid Amplification byly zaznamenány u 78 nemocných (78 %). Mikrometastázy v lymfatických uzlinách byly metodou One-Step Nucleic Acid Amplification při negativitě ostatních metod prokázány u 16 pacientů (16 %). Pouze ve 3 případech (3 %) bylo vyšetření hematoxylinem-eosinem, resp. imunohistochemií s protilátkou proti cytokeratinu 19 pozitivní při negativitě metody One-Step Nucleic Acid Amplification. Výsledky imunohistochemie s protilátkou proti cytokeratinu 19 prakticky přesně kopírovaly výsledky vyšetření hematoxylinem-eosinem (97 %). Závěr: Výsledky studie prokázaly vyšší procento detekovaných mikrometastáz v hilových a mediastinálních lymfatických uzlinách při vyšetření metodou One-Step Nucleic Acid Amplification ve srovnání s hematoxylinem-eosinem a imunohistochemií s protilátkou proti cytokeratinu 19 (upstaging 16 %). Ukazuje se tak, že vyšetření lymfatických uzlin pomocí metody One-Step Nucleic Acid Amplifi cation může mít určitý potenciál zpřesnit staging plicních nádorů, naopak imunohistochemie s protilátkou proti cytokeratinu 19 změny stagingu téměř nepřináší. Je však nezbytné potvrdit tento předpoklad v dalších studiích, resp. na větším souboru nemocných. Dalším úkolem je také pečlivým sledováním nemocných zjistit souvislost mikrometastáz detekovaných v lymfatických uzlinách metodou One-Step Nucleic Acid Amplification s jejich follow-up.

Introduction: The aim of this article is to compare the sensitivity of detecting micrometastases in hilar and mediastinal lymph nodes in case of primary (non-small cell) and secondary (metastases of colorectal carcinoma) pulmonary tumours using standard histopathological examination with haematoxylin-eosin staining, immunohistochemistry examination with Anti-Cytokeratin 19 antibody and examination based on the One-Step Nucleic Acid Amplification method. Method: During radical surgical treatment of primary non-small cell lung carcinoma and pulmonary metastases of colorectal carcinoma, hilar and mediastinal lymph nodes of 100 patients enrolled in the study in the period from 2015 to 2017 were extracted based on a standard classifi cation. These lymph nodes were subsequently divided along the longitudinal axis into 4 identical parts where part one and three on the left were intended for examination based on the One-Step Nucleic Acid Amplification method, whereas parts two and four were subjected to histopathological examination. In evaluating the respective parts of the nodes by histological examination, the nodes were fi rst examined by a standard procedure that involves haematoxylin-eosin staining, followed by immunohistochemistry examination with Anti-Cytokeratin 19 antibody. The One-Step Nucleic Acid Amplification method was performed in the kit supplied by Sysmex (Kobe, Japan) and is based on the detection of cytokeratin 19 mRNA (messenger ribonucleic acid) by reverse transcription coupled with isothermal amplifi cation. Results: A total of 1,426 lymph nodes of the patients enrolled in the study were extracted and examined using the above mentioned methodology. In 78 patients (78%), identical results were obtained using haematoxylin-eosin staining, immunohistochemistry with Anti-Cytokeratin 19 and One- Step Nucleic Acid Amplifi cation. Micrometastases in the lymph nodes using the One-Step Nucleic Acid Amplifi cation method in the absence of the other methods were proven in 16 patients (16%). Only in 3 cases (3%), the examination by haematoxylin-eosin staining, or immunohistochemistry with Anti-Cytokeratin 19, was positive while One-Step Nucleic Acid Amplification was negative. The results obtained by immunohistochemistry with Anti-Cytokeratin 19 antibody were practically the same as those obtained by haematoxylin-eosin staining (97%). Conclusion: The results of the study have demonstrated a higher percentage of metastases detected in hilar and mediastinal lymph nodes if the One-Step Nucleic Acid Amplification method of examination was used compared to haematoxylin-eosin staining and immunohistochemistry with Anti-Cytokeratin 19 antibody (upstaging in 16%). This shows that the examination of lymph nodes using the One-Step Nucleic Acid Amplification method can have a certain potential to make the pulmonary tumours staging more accurate. On the other hand, immunohistochemistry with Anti- Cytokeratin 19 antibody seems to be not so useful. However, it is necessary to prove this hypothesis in follow-up studies, or where applicable, in a larger cohort of patients. Another task is to ascertain, by careful patient monitoring, the infl uence of the micrometastases detected in their lymph nodes using the One-Step Nucleic Acid Amplification method on these patients’ follow-up.

• MeSH
• barvení a značení MeSH
• hematoxylin terapeutické užití   MeSH
• imunohistochemie MeSH
• keratin-19 terapeutické užití   MeSH
• lidé MeSH
• lymfadenektomie MeSH
• lymfatické uzliny * anatomie a histologie   diagnostické zobrazování   MeSH
• metastázy nádorů diagnóza   terapie   MeSH
• nádory plic * diagnóza   chirurgie   MeSH
• prospektivní studie MeSH
• techniky amplifikace nukleových kyselin metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem 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

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521 bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500 pg/μL. DNA concentration as low as 10 pg/μL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100 pg/μL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

• MeSH
• alkoholoxidoreduktasy * genetika   MeSH
• Bacillus subtilis * genetika   izolace a purifikace   enzymologie   MeSH
• bakteriální proteiny genetika   MeSH
• diagnostické techniky molekulární * metody   MeSH
• kolorimetrie * metody   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• půdní mikrobiologie MeSH
• senzitivita a specificita MeSH
• techniky amplifikace nukleových kyselin * metody   MeSH
• Publikační typ
• časopisecké články MeSH