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1.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 801-809, 2020 Apr 25.
Artículo en Chino | MEDLINE | ID: mdl-32347074

RESUMEN

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.


Asunto(s)
Escherichia coli , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Cartilla de ADN/genética , Mutagénesis Sitio-Dirigida/métodos , Plásmidos/genética
2.
Int J Mol Sci ; 21(8)2020 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-32344568

RESUMEN

Testing for the presence of coronavirus is an essential diagnostic tool for monitoring and managing the current COVID-19 pandemic. The only reliable test in current use for testing acute infection targets the genome of SARS-CoV-2, and the most widely used method is quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR). Despite its ubiquity, there is a significant amount of uncertainty about how this test works, potential throughput and reliability. This has resulted in widespread misrepresentation of the problems faced using this test during the current COVID-19 epidemic. This primer provides simple, straightforward and impartial information about RT-qPCR.


Asunto(s)
Betacoronavirus/aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Cartilla de ADN , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Betacoronavirus/genética , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Pandemias , Neumonía Viral/diagnóstico , Neumonía Viral/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación
3.
Rev Med Virol ; 30(3): e2106, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32302058

RESUMEN

Emerging and reemerging infectious diseases are global public concerns. With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus, SARS-CoV-2 has been attracting tremendous attention. Rapid and accurate laboratory testing of SARS-CoV-2 is essential for early discovery, early reporting, early quarantine, early treatment, and cutting off epidemic transmission. The genome structure, transmission, and pathogenesis of SARS-CoV-2 are basically similar to SARS-CoV and MERS-CoV, the other two beta-CoVs of medical importance. During the SARS-CoV and MERS-CoV epidemics, a variety of molecular and serological diagnostic assays were established and should be referred to for SARS-CoV-2. In this review, by summarizing the articles and guidelines about specimen collection, nucleic acid tests (NAT) and serological tests for SARS-CoV, MERS-CoV, and SARS-CoV-2, several suggestions are put forward to improve the laboratory testing of SARS-CoV-2. In summary, for NAT: collecting stool and blood samples at later periods of illness to improve the positive rate if lower respiratory tract specimens are unavailable; increasing template volume to raise the sensitivity of detection; putting samples in reagents containing guanidine salt to inactivate virus as well as protect RNA; setting proper positive, negative and inhibition controls to ensure high-quality results; simultaneously amplifying human RNase P gene to avoid false-negative results. For antibody test, diverse assays targeting different antigens, and collecting paired samples are needed.


Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Enfermedades Transmisibles Emergentes/virología , Anticuerpos Antivirales/aislamiento & purificación , Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , Enfermedades Transmisibles Emergentes/diagnóstico , Infecciones por Coronavirus/diagnóstico , Cartilla de ADN , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Ribonucleasa P/genética , Ribonucleasa P/aislamiento & purificación , Virus del SRAS/genética , Virus del SRAS/aislamiento & purificación , Pruebas Serológicas/métodos
4.
Parasitol Res ; 119(3): 1173-1176, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32140779

RESUMEN

Babesia is tick-transmitted protozoan parasites that infect mammalian hosts and have a major impact on farm and pet health-associated costs worldwide. This study aimed to test the prevalence of Babesia spp. infection in a small cohort of dogs at a veterinary hospital and to perform molecular characterization of the Babesia species causing the infection. For the PCR assay, 5 mL of blood was collected by venipuncture of the cephalic or radial veins in 300 dogs of different ages, sex, and breeds, which were presented to the veterinary hospital of the Federal University of Uberlândia between March 2015 and April 2016. In addition, a drop of blood was collected from the marginal blood vessels of the ear of dogs included in this study. Ninety-two (30.67%) were positive for Babesia spp., as determined by microscopic observation of the blood smear, revealing the presence of intra-erythrocyte merozoites. For molecular characterization by PCR, 17 samples were chosen from dogs who were tested positive for Babesia spp. by blood smears. Among them, B. vogeli was found to infect all 17 dogs, as determined by 99-100% sequence identity (closest GenBank match KT246307) using primers PIRO A/PIRO B. Our results indicate that the species observed in these dogs was B. vogeli.


Asunto(s)
Babesiosis/parasitología , Enfermedades de los Perros/parasitología , Animales , Babesia/genética , Brasil/epidemiología , Cartilla de ADN , Perros , Femenino , Masculino , Reacción en Cadena de la Polimerasa/veterinaria , Prevalencia , Garrapatas/parasitología
5.
Plant Dis ; 104(5): 1514-1526, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32105572

RESUMEN

Sclerotinia sclerotiorum is one of the most devastating and cosmopolitan plant pathogens. Rapid detection of S. sclerotiorum can provide growers an advantage in knowing what control measures should be taken to minimize crop damage and financial losses caused by it. Loop-mediated isothermal amplification (LAMP) is a fast, sensitive, and specific nucleic acid amplification method that does not require a thermal cycler. This study aimed to develop a LAMP-based assay for the specific detection of S. sclerotiorum (Ss-LAMP). A real-time quantitative LAMP reaction (Ss-qLAMP) and a calcein ion indicator-based LAMP reaction (Ss-cLAMP) were designed, optimized, and tested on fungi, plant, and soil samples. The Ss-LAMP reactions were very specific and sensitive. Applying the artificially inoculated soil samples with DNA purified by five protocols in the Ss-qLAMP reaction, it was possible to detect and quantify the pathogen DNA, regardless of the extraction protocol. Naturally infected soybean tissues had the pathogen detected by Ss-cLAMP directly in the reaction tube with no DNA extraction requirement. The assays should be applicable for many types of samples, such as soil, spore traps, and plant tissues from several crops, with no requirement for DNA extraction. The Ss-LAMP reactions took less than 1 h to complete, and they can be made directly in the field with real-time quantitative results (Ss-qLAMP) or qualitative naked-eye visual results (Ss-cLAMP). Results were obtained with 10 pg of DNA or 10 ng of crude mycelium, suggesting a detection limit close to a single DNA copy. Ss-LAMP reactions will allow rapid and accurate diagnosis of S. sclerotiorum and assist in pathogen management and control.


Asunto(s)
Ascomicetos , Técnicas de Amplificación de Ácido Nucleico , Cartilla de ADN , Metales , Reacción en Cadena de la Polimerasa
6.
Arch Virol ; 165(3): 743-747, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31980939

RESUMEN

A cross-priming isothermal amplification (CPA) assay was developed for detection of feline herpesvirus type 1 (FHV-1). In this assay, the target fragment of the FHV-1 glycoprotein B gene is amplified rapidly by Bst DNA polymerase at a constant temperature (63 °C, 45 min), using a simple thermostat. The assay had no cross-reactions with four types of feline viruses, and the detection limit was 100 copies/µl. The positive rate of clinical samples from CPA was 100% consistent with qPCR but higher than ordinary PCR, indicating its superiority to ordinary PCR. Visualization was achieved using SYBR Green I dye.


Asunto(s)
Enfermedades de los Gatos/virología , Reactividad Cruzada , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Varicellovirus/aislamiento & purificación , Proteínas del Envoltorio Viral/aislamiento & purificación , Animales , Enfermedades de los Gatos/diagnóstico , Gatos , Cartilla de ADN/genética , Técnicas de Amplificación de Ácido Nucleico/economía , Técnicas de Amplificación de Ácido Nucleico/métodos , Sensibilidad y Especificidad
7.
Food Microbiol ; 87: 103367, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-31948615

RESUMEN

Listeria monocytogenes is an important foodborne pathogen, causative agent of listeriosis. The epidemiology and persistence of this bacterium in meat processing plants may be related to its serotype, so it is of utmost importance to carry out a correct differentiation of L. monocytogenes serotypes. The objective of this study was to develop a unique quadruplex real-time quantitative PCR (qPCR) method able to differentiate the four most predominant and worrying L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) in isolates from meat processing plants and ready-to-eat (RTE) dry-cured meat products. The design of specific primers and probes was based on the lmo0737, lmo0308, ORFC (locus genomically equivalent to gltA-gltB) and ORF2110 genes. A qPCR based on a fragment of the 16S rRNA gene was used to ensure the amplification of Listeria spp. genomic DNA. The standard curves showed efficiency values ranging between 92.3% and 105.8% and, R2 values > 0.98. The specificity of the method was also confirmed by the comparison of the results with those obtained by a previously reported conventional multiplex PCR. In addition, none of the strains which were not ascribed to L. monocytogenes amplified any of the target genes related to the four major serotypes of this pathogenic species. The qPCR, therefore, provides a sensitive, specific and rapid tool for identifying the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c and 4b. This method could be very useful for identifying sources of L. monocytogenes contamination in the meat industry or for epidemiological monitoring of persistent strains throughout the processing of RTE meat products.


Asunto(s)
Listeria monocytogenes/aislamiento & purificación , Carne/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Cartilla de ADN/genética , ADN Bacteriano/genética , Contaminación de Alimentos/análisis , Manipulación de Alimentos/instrumentación , Listeria monocytogenes/clasificación , Listeria monocytogenes/genética , Productos de la Carne/microbiología , ARN Ribosómico 16S/genética , Serogrupo
8.
J Forensic Sci ; 65(1): 283-287, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31436852

RESUMEN

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.


Asunto(s)
Escarabajos/genética , Complejo IV de Transporte de Electrones/genética , Análisis de Secuencia de ADN , Animales , Cartilla de ADN , Larva , Filogenia , Reacción en Cadena de la Polimerasa , República de Corea , Especificidad de la Especie
9.
Arch Microbiol ; 202(1): 171-179, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31549205

RESUMEN

Alternaria leaf spot (ALS) caused by Alternaria carthami Chowdhary is one of the major threats to the cultivation of safflower in the world. The pathogen is seed borne and requires early detection for restricting its transmission and proliferation. A PCR-based diagnostic assay was developed for easy, quick and reliable detection of A. carthami in infected seeds and leaf samples of safflower. A primer set, AcSPF and AcSPR was designed using ribosomal internal transcribed spacer regions of A. carthami that consistently produced a distinct amplicon of 340 bp with DNA extracted from thirty A. carthami isolates. The specificity of the primer was confirmed using strains of 26 other strains of Alternaria and four other fungal pathogens of safflower. The sensitivity of detection was further enhanced from concentration of 100 pg by simple PCR to as low as 10 pg fungal DNA by a nested PCR assay using ITS and AcSPF and AcSPR primers. The primer pair also facilitated detection of A. carthami in infected seeds and leaf samples. The study provides an accurate and sensitive diagnostic tool for detection of A. carthami.


Asunto(s)
Agricultura/métodos , Alternaria/genética , Reacción en Cadena de la Polimerasa , Carthamus tinctorius/microbiología , Cartilla de ADN/genética , ADN de Hongos/genética , Hojas de la Planta/microbiología , Reacción en Cadena de la Polimerasa/normas , Semillas/microbiología , Sensibilidad y Especificidad
10.
J Forensic Sci ; 65(1): 52-60, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31433500

RESUMEN

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used "legal high" opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta-D-glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high-resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/µL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen® PLUS.


Asunto(s)
Cannabis/genética , ADN de Plantas/genética , Toxicología Forense/métodos , Mitragyna/genética , Cartilla de ADN , Electroforesis en Gel de Agar , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Temperatura de Transición
11.
Anal Bioanal Chem ; 412(1): 93-101, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31797016

RESUMEN

The aim of this study was to develop an effective and specific visual method to rapidly detect and identify Vibrio parahaemolyticus (V. parahaemolyticus) based on the polymerase spiral reaction (PSR). The method utilized only two pairs of primers designed specifically to target the conserved tlh gene sequence of V. parahaemolyticus. Nucleic acid amplification can be achieved under isothermal conditions using DNA polymerase. The reaction could be accomplished in < 40 min with high specificity and sensitivity. The limits of detection of V. parahaemolyticus in purified genomic DNA and pure culture were 300 fg/µL and 2.4 CFU/mL per reaction, respectively, which were 100-fold more sensitive than with conventional PCR. The model food samples showed consistent specificity and sensitivity to the pure bacterial culture. With these encouraging results, it is expected that the novel, effortless and reliable isothermal nucleic acid testing assay developed in this study has potential to be applied to screening for V. parahaemolyticus in seafood samples.


Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Vibrio parahaemolyticus/aislamiento & purificación , Cartilla de ADN , ADN Bacteriano/análisis , Genes Bacterianos , Límite de Detección , Vibrio parahaemolyticus/genética
12.
J Sci Food Agric ; 100(4): 1687-1693, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-31803942

RESUMEN

BACKGROUND: The growth of halal food consumption worldwide has resulted in an increase in the request for halal authentication. DNA-based detection using powerful real-time polymerase chain reaction (PCR) technique has been shown to be highly specific and sensitive authentication tool. The efficient DNA extraction method in terms of quality and quantity is a backbone step to obtain successful real-time PCR assays. In this study, different DNA extraction methods using three lysis buffers were evaluated and developed to recommend a much more efficient method as well as achieve a successful detection using real-time PCR. RESULTS: The lysis buffer 2 (LB2) has been shown to be the best lysis buffer for DNA extraction from both raw and processed meat samples comparing to other lysis buffers tested. Hence, the LB2 has been found to be ideal to detect meat and porcine DNAs by real-time PCR using pairs of porcine specific primers and universal primers which amplified at 119 bp fragment and 93 bp fragment, respectively. This assay allows detection as low as 0.0001 ng of DNA. Higher efficiency and sensitivity of real-time PCR via a simplified DNA extraction method using LB2 have been observed, as well as a reproducible and high correlation coefficient (R2  = 0.9979) based on the regression analysis of the standard curve have been obtained. CONCLUSION: This study has established a fast, simple, inexpensive and efficient DNA extraction method that is feasible for raw and processed meat products. This extraction technique allows an accurate DNA detection by real-time PCR and can also be implemented to assist the halal authentication of various meat-based products available in the market. © 2019 Society of Chemical Industry.


Asunto(s)
Métodos Analíticos de la Preparación de la Muestra/métodos , ADN/genética , ADN/aislamiento & purificación , Contaminación de Alimentos/análisis , Productos de la Carne/análisis , Animales , Búfalos/genética , Bovinos , Pollos/genética , Cartilla de ADN/genética , Patos/genética , Cabras/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Porcinos/genética , Pavos/genética
13.
Biosci Biotechnol Biochem ; 84(1): 43-52, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31495297

RESUMEN

To date, studies on the application of loop-mediated isothermal amplification (LAMP) in the detection of genetically modified organisms (GMOs) are stably increasing and demonstrates LAMP is a potential and promising method for on spot identification of GMOs. However, little information is known for detection of GM potato events by LAMP. In this report, we developed an optimized and visual LAMP assay with high specificity and sensitivity to rapidly amplify genomic DNA of potato EH92-527-1 within 45 min. The limit of detection of LAMP in our study is 10-fold higher than the conventional PCR. Furthermore, LAMP products can be directly observed via naked eyes by addition of SYBR Green I without gel electrophoresis analysis and PCR-based equipment. Therefore, the LAMP assay developed in this paper provides an efficient, convenient and cost-effective tool for the detection of GM potato EH92-527-1.


Asunto(s)
ADN de Plantas/genética , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Solanum tuberosum/genética , Secuencia de Bases/genética , Percepción de Color , Cartilla de ADN/genética , Enzimas de Restricción del ADN/genética , Electroforesis en Gel de Agar , Colorantes Fluorescentes/química , Contaminación de Alimentos/análisis , Amplificación de Genes , Límite de Detección , Compuestos Orgánicos/química , Reacción en Cadena de la Polimerasa/economía , Sensibilidad y Especificidad , Temperatura , Tiempo
14.
Insect Sci ; 27(1): 143-158, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29873880

RESUMEN

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.


Asunto(s)
Código de Barras del ADN Taxonómico/métodos , Cartilla de ADN/análisis , Tephritidae/clasificación , Animales , Asia Sudoriental , Australia , Secuencia de Bases , Complejo IV de Transporte de Electrones/análisis , Proteínas de Insectos/análisis , Masculino , Islas del Pacífico , Filogenia , Tephritidae/genética
15.
Food Microbiol ; 86: 103310, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31703859

RESUMEN

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.


Asunto(s)
Antibacterianos/farmacología , Azidas/farmacología , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/crecimiento & desarrollo , Propidio/análogos & derivados , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Cartilla de ADN/genética , ADN Bacteriano/genética , Desecación , Calor , Listeria monocytogenes/química , Listeria monocytogenes/genética , Viabilidad Microbiana/efectos de los fármacos , Propidio/farmacología
16.
Food Chem ; 310: 125955, 2020 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-31841941

RESUMEN

Vibrio parahaemolyticus is a major hidden danger of food safety. To develop a rapid, sensitive and on-site detecting method of Vibrio parahaemolyticus (V. parahaemolyticus), a strand replacement primer thermostat phosphate (SRPP) visual sensor was proposed, based on Bst DNA polymerase and pyrophosphatase. The novel strand replacement primer (SRP) facilitates chain substitution and to open a self-folding hairpin by adding region at its 3' end. Under the action of the SRP, a pair of external primers and two inner primers, target DNA is specifically amplified at 63 °C relies mainly on the hairpin. Many pyrophosphates (PPi) are simultaneously generated as by-products, which can be converted into phosphates (Pi) by pyrophosphatase for phosphomolybdate blue visual detection within 5 min. The proposed biosensor can detect 1.29 × 103 copies of V. parahaemolyticus within 35 min.


Asunto(s)
Cartilla de ADN/genética , Análisis de los Alimentos/instrumentación , Microbiología de Alimentos/métodos , Vibrio parahaemolyticus/genética , ADN Polimerasa Dirigida por ADN/genética , Análisis de los Alimentos/métodos , Pirofosfatasas/genética , Sensibilidad y Especificidad
17.
Food Microbiol ; 85: 103304, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31500716

RESUMEN

High-resolution melting (HRM) analysis followed by sequencing was applied for determination of bacteria grown on plates isolated from farmed mussels (Mytilus galloprovincialis) during their storage at 4 °C. The V3-V4 region of the 16S rRNA gene from the isolates was amplified using 16S universal primers. Melting curves (peaks) and high resolution melting curves (shape) of the amplicons and sequencing analysis were used for differentiation and identification of the isolated bacteria, respectively. The majority of the isolates (a sum of 101 colonies, from five time intervals: day 0, 2, 4, 6 and 8) from non-selective solid medium plates were classified in four bacterial groups based on the melting curves (peaks) and HRM curves (shape) of the amplicons, while three isolates presented distinct HRM curve profiles (single). Afterwards, sequencing analysis showed that the isolates with a) the same melting peak temperature and b) HRM curves that were >95% similar grouped into the same bacterial species. Therefore, based on this methodology, the cultivable microbial population of chill-stored mussels was initially dominated by Psychrobacter alimentarius against others, such as Psychrobacter pulmonis, Psychrobacter celer and Klebsiella pneumoniae. P. alimentarius was also the dominant microorganism at the time of the sensory rejection (day 8). Concluding, HRM analysis could be used as a useful tool for the rapid differentiation of the bacteria isolated from mussels during storage, at species level, and then identification is feasible by the sequencing of one only representative of each bacterial species, thus reducing the cost of required sequencing.


Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , Bivalvos/microbiología , Almacenamiento de Alimentos , Refrigeración , Animales , Cartilla de ADN/genética , ARN Ribosómico 16S/genética
18.
Clin Lab ; 65(12)2019 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-31850703

RESUMEN

BACKGROUND: In duplex real time quantitative PCR (qPCR), there are several factors affecting the sensitivity of duplex qPCR, such as sharing primer, quantity of the internal control (IC) gene, and the primer dimer (PD). The aim of the study is to find out the relationship of interference among templates with different primer pairs, the internal control gene, and the PD in duplex PCR. METHODS: We designed and synthesized plasmids with partial same sequence and different types of primers include central-homo primer pair, ordinary primer pair, and complementary primer pair. Then we compared the amplification efficiencies of different kinds of primer pairs. Besides, we adjusted the amount of IC plasmid and IC primer to find out the key factor that influences the sensitivity of the target template. RESULTS: The concentration ratios of two plasmids showed interference in sharing the universal primer pair, sharing one forward primer, and sharing no primer were 50:1, 200:1 and 500:1, respectively. The amplification efficiency of the ordinary primer pair was higher than that of the universal primer pair for the plasmid. Sensitivity of the duplex qPCR remained unchanged when the amount of PDs increased, but it declined rapidly when the quantity of the IC increased. CONCLUSIONS: IC is the major factor influencing the sensitivity of the duplex qPCR and it would be better to use one universal primer for IC and target template to achieve minimum interference.


Asunto(s)
Cartilla de ADN/genética , Regulación de la Expresión Génica , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Secuencia de Bases , ADN Viral/genética , Hepatitis B/diagnóstico , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
19.
BMC Biotechnol ; 19(1): 99, 2019 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-31856784

RESUMEN

BACKGROUND: To avoid destructive sampling for conservation and genetic assessment, we isolated the DNA of clam Cyclina sinensis from their feces. DNA electrophoresis and PCR amplification were used to determine the quality of fecal DNA. And we analyzed the effects of different conditions on the degradation of feces and fecal DNA. RESULTS: The clear fecal DNA bands were detected by electrophoresis, and PCR amplification using clam fecal DNA as template was effective and reliable, suggesting that clam feces can be used as an ideal material for noninvasive DNA isolation. In addition, by analyzing the effects of different environmental temperatures and soaking times on the degradation of feces and fecal DNA, we found that the optimum temperature was 4 °C. In 15 days, the feces maintained good texture, and the quality of fecal DNA was good. At 28 °C, the feces degraded in 5 days, and the quality of fecal DNA was poor. CONCLUSIONS: The clam feces can be used as an ideal material for noninvasive DNA isolation. Moreover, the quality of fecal DNA is negatively correlated with environmental temperature and soaking time.


Asunto(s)
Bivalvos/genética , ADN/genética , Heces/química , Animales , ADN/aislamiento & purificación , Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa
20.
Invest Ophthalmol Vis Sci ; 60(14): 4904-4914, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31770435

RESUMEN

Purpose: Uveal melanoma is a common primary intraocular malignancy accompanied by high mortality. Previous evidence has highlighted the implication of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in uveal melanoma. Accordingly, we further uncovered the possible role of lncRNA plasmacytoma variant translocation 1 gene (PVT1) and microRNA-17-3p (miR-17-3p) in uveal melanoma. Methods: A series of experiments were performed to examine the relationship among lncRNA PVT1, miR-17-3p, and murine double minute clone 2 oncoprotein (MDM2). Afterward, gain- and loss-of-function approaches were used with uveal melanoma cells to verify the role of lncRNA PVT1, miR-17-3p, and MDM2 in the tumorigenesis and development of uveal melanoma. Results: Highly expressed lncRNA PVT1 and MDM2, yet lowly expressed miR-17-3p, were identified in ocular uveal melanoma tissues versus normal adjacent tissues. Then, dual luciferase reporter gene assay, RNA binding protein immunoprecipitation, and RNA pull-down assays showed that lncRNA PVT1 specifically bound to miR-17-3p, and that MDM2 was a target gene of miR-17-3p. Gain- and loss-of-function studies elucidated that silencing of lncRNA PVT1 or overexpression of miR-17-3p resulted in decreased MDM2 expression and increased transcriptional activity of p53, in addition to inhibiting uveal melanoma cell proliferation, migration, and invasion, yet promoted cell apoptosis in vitro. In addition, lncRNA PVT1 silencing or miR-17-3p overexpression was noted to inhibit tumor growth in vivo. Conclusions: Downregulation of lncRNA PVT1 could potentially promote miR-17-3p expression to suppress tumorigenesis and development of uveal melanoma by activating the p53 signaling pathway through binding to MDM2.


Asunto(s)
Silenciador del Gen/fisiología , Melanoma/prevención & control , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Largo no Codificante/genética , Neoplasias de la Úvea/prevención & control , Animales , Western Blotting , Movimiento Celular/fisiología , Supervivencia Celular , Cartilla de ADN/química , Técnica del Anticuerpo Fluorescente Indirecta , Genes Reporteros , Hibridación Fluorescente in Situ , Masculino , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Activación Transcripcional , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Neoplasias de la Úvea/metabolismo
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