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1.
Mikrobiyol Bul ; 53(4): 442-450, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709941

RESUMO

Toxoplasmosis is one of the most common parasitic infection in humans. Serological and molecular methods are used for diagnosis. Molecular methods are becoming increasingly preferred, since they lead to shortening of diagnostic time. In our study, it was aimed to determine Toxoplasma gondii by a cost-effective, quantitative, fast and reliable method without using a commercial kit, and apply method verification. T.gondii strain which was continued by mouse inoculation in our laboratory was used for method verification study. For this purpose DNA extraction was performed using a commercial kit. The limit of detection and, high and low positivity rates were determined by serial dilutions of DNA sample. Accuracy and certainty studies were performed using with TG-F, TG-R primers and TaqMan TG probe for method verification of the test. In the study with serial dilutions of DNA sample, detection limit was determined as 10-3 dilutions (0.028 copies/reaction). Furthermore 10-1 dilution (2.8 copies/reaction) was considered as high positive, 10-2 dilution (0.28 copies/reaction) was considered as low positive and method verification studies were performed. The accuracy of test was determined as 0.62 for high positive samples and 0.14 for low positive samples. CV value of intra-assay certainty was 0.62 for high positive samples and 0.14 for low positive samples, whereas, CV value of inter-assay certainty was calculated as 1.03 for high positive samples and 2.34 for low positive samples. Correlation coefficient was determined as 0.99. The coefficient of variation of inhouse realtime PCR method used in our study was found to be below 15%, and it was decided to be suitable for routine laboratory studies.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real , Toxoplasma , Toxoplasmose , Animais , Primers do DNA , DNA de Protozoário , Humanos , Camundongos , Parasitologia/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Toxoplasma/genética , Toxoplasmose/diagnóstico
2.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(5): 468-473, 2019 Oct 16.
Artigo em Chinês | MEDLINE | ID: mdl-31713373

RESUMO

OBJECTIVE: To establish a recombinase aided isothermal amplification (RAA) assay for detection of Clonorchis sinensis. METHODS: The 18S ribosomal RNA (18S rRNA) sequence of C. sinensis was used as the target sequence, and specific primers and probes were designed, synthesized and screened to establish a rapid fluorescent RAA assay for the detection of C. sinensis. Then, the sensitivity of the fluorescent RAA assay was evaluated using the recombinant plasmids containing various copy numbers of DNA fragments and C. sinensis genomic DNA at various concentrations, and the specificity of the fluorescent RAA as say was evaluated using the genomic DNA of Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale and S. mansoni as templates. DNA samples were extracted from the feces containing C. sinensis eggs and freshwater fish containing metacercaria for the fluorescent RAA assay, and the performance for detection of C. sinensis-infected samples was preliminarily assessed in the field. RESULTS: A fluorescent RAA assay for detection of C. sinensis was successfully established, which was feasible for specific amplification of C. sinensis genomic DNA at 39 °C within 20 min. The lowest detection limit was 10 copies/µL if the recombinant plasmid containing various copy numbers of DNA fragments was used as a template, and the lowest detection limit was 3 pg/µL if the C. sinensis genomic DNA at various concentrations served as a template. All detections were negative if the genomic DNA of A. lumbricoides, E. granulosus, S. japonicum, A. duodenale, and S. mansoni was used as templates. In addition, the fluorescent RAA assay showed a high performance for the detection of C. sinensis-infected samples in the field, which successfully detected C. sinensis-infected human and rat fecal samples and Pseudorasbora parva samples. CONCLUSIONS: A fluorescent RAA assay is successfully established, which is simple, rapid, sensitivity and specific for detection of C. sinensis.


Assuntos
Clonorquíase , Clonorchis sinensis , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos , Animais , Clonorquíase/diagnóstico , Clonorchis sinensis/genética , Primers do DNA , Fezes/parasitologia , Humanos , Limite de Detecção , Parasitologia/métodos , Ratos , Recombinases/metabolismo , Sensibilidade e Especificidade
3.
Undersea Hyperb Med ; 46(5): 635-646, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31683362

RESUMO

We aimed to assess the effects of intermittent hyperbaric oxygenation (HBO2 at 2 bars for 120 minutes a day for four successive days) on acetylcholine-induced vasorelaxation (AChIR) in female Sprague-Dawley (SD) rats (N=80) that were randomized into four groups: healthy controls (CTR); diabetic rats (DM); and control and diabetic rats that underwent hyperbaric oxygenation (CTR+HBO and DM+HBO), respectively. AChIR was measured in vitro in aortic rings, with/without L-NAME, MS-PPOH, HET0016 or indomethacin. mRNA expression of eNOS, iNOS, COX-1, COX-2, thromboxane A synthase 1 (TBXAS1), CYP4A1, CYP4A3 and CYP2J3 was assessed by qPCR. Systemic oxidative stress and plasma antioxidative capacity were determined with the thiobarbituric acid-reactive substances (TBARS) and the ferric reducing ability of plasma (FRAP) assays, respectively. There was no significant difference in AChIR among experimental groups of rats. In CTR and DM group of rats, AChIR was mediated by NO and EETs pathway, while in the CTR+HBO and DM+HBO groups, NO-pathway prevailed. iNOS expression was upregulated in the DM group compared to CTR, while HBO2 upregulated eNOS in CTR group and TBXAS1 in DM group of rats. In both, CTR and DM group of rats, the sensitivity to ACh in the presence of L-NAME or in the presence of MSPPOH was significantly decreased compared to the response to ACh in the absence or presence of indomethacin or HET0016. DM and DM+HBO rats had increased TBARS compared to their respective controls. In conclusion, HBO2 presumably alters vasorelaxation in response to ACh from NO-EETs mediated pathways to solely NO-pathway, without affecting oxidative status of DM rats.


Assuntos
Acetilcolina/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Oxigenação Hiperbárica , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Análise de Variância , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Glicemia/análise , Peso Corporal , Sistema Enzimático do Citocromo P-450/fisiologia , Primers do DNA , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Oxigenação Hiperbárica/métodos , Estresse Oxidativo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Estreptozocina , Fatores de Tempo , Vasodilatação/fisiologia
4.
Enzymes ; 45: 289-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627881

RESUMO

PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn2+ ions to stabilize the interaction of the incoming deoxynucleotide at the 3'-site. The capacity to bind both ssDNA template and 3'-deoxynucleotide was shown to reside in the AEP core of PrimPol, with ZnFD being dispensable at these two early steps of the primase reaction. Sugar selection favoring dNTPs versus NTPs at the 3' site is mediated by a specific tyrosine (Tyr100) acting as a steric gate. Besides, a specific glutamate residue (Glu116) conforming a singular A motif (DxE) promotes the use of Mn2+ to stabilize the pre-ternary complex. Mirroring the function of the PriL subunit of dimeric AEP primases, the ZnFD of PrimPol is crucial to stabilize the initiating 5'-nucleotide, specifically interacting with the gamma-phosphate. Such an interaction is crucial to optimize dimer formation and the subsequent translocation events leading to the processive synthesis of a mature DNA primer. Finally, the capacity of PrimPol to tolerate lesions is discussed in the context of its DNA primase function, and its potential as a TLS primase.


Assuntos
DNA Primase/metabolismo , Primers do DNA/biossíntese , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Humanos
5.
Exp Parasitol ; 207: 107773, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31605671

RESUMO

Studies of the primers that were designed to detect New World Leishmania were systematically reviewed to report the characteristics of each target, detection limit, specificity of the primers designed and diagnostic sensibility. The papers identified in the databases PubMed and Web of Science involved 50 studies. Minicircle is the most applied target in molecular research for diagnosis, due to its high sensitivity in detecting Leishmania in different clinical samples, a characteristic that can be partially attributed to the higher number of copies of the minicircle per cell. The other molecular targets shown in this review were less sensitive to diagnostic use because of the lower number of copies of the target gene per cell, but more specific for identification of the subgenus and/or species. The choice of the best target is an important step towards the result of the research. The target allows the design of primers that are specific to the genus, subgenus or a particular species and also imparts sensitivity to the method for diagnosis. The findings of this systematic review provide the advantages and disadvantages of the main molecular targets and primers designed for New World Leishmania, offering information so that the researcher can choose the PCR system best suited to their research need. This is a timely and extremely thorough review of the primers designed for New World Leishmania.


Assuntos
Primers do DNA/análise , DNA de Protozoário/análise , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da Polimerase/métodos , Humanos , Leishmania/isolamento & purificação , Limite de Detecção , Sensibilidade e Especificidade
6.
Klin Lab Diagn ; 64(9): 560-564, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31610109

RESUMO

Human babesiosis caused by parasitic protozoan Babesia spp. is sporadic zoonotic vector-borne infection. The course of babesiosis and prognosis depend on the type of pathogen and on the patient's immunological status. Significance this disease is a severe, often fatal course with immunocompromissed patients resembling complicated falciparum malaria. In Europe to date, more than 50 cases of confirmed human babesiosis have been reported in most cases caused by Babesia divergens. Possible there are unrecognized cases. Pathogen is an obligate intraerythrocyte parasite of vertebrate animals. The organism is transmitted from animal to man through bite of Ixodidae tick. Asexual reproduction of the parasite occurs in a vertebrate host. The pathogenesis of babesiosis is caused by the destruction of host cells. Intensive haemolysis of red blood cells leads to the development of haemolytic anemia, haematuria, jaundice, and polyorgan failure may develop. The clinical manifestations of the disease are nonspecific. Detection of intraerythrocyte parasites in blood smears stained Gimsa-Romanovsky confirms the proposed diagnosis. Blood smears and some laboratory signs from fatal cases were analyzed in the Reference-centre of E. I. Martsinovskii Institute. Original microphotographs B. divergens are shown. The main morphological forms of the parasite are shown. In addition to the well-known tetrades of parasites «Maltese Cross¼, for the first time, the parasites dividing into 6 interconnected trophozoites - "sextet" - were found. Originally, the invasion of Babesia in a normoblast is shown. An unusually high multiple invasion (14 parasites) of erythrocytes is noted. Because the patients, initially, were incorrectly diagnosed with malaria, the differential diagnosis of Babesia with Plasmodium is described step-by-step. It is important, since the treatment with antimalarial drugs is ineffective. Deviation laboratory signs are discussed. Complex morphological characteristics allowed us to speciated the parasites as B. divergens. DNA was detected in the sample with specific primers Bab di hsp70F/Bab di hsp70R and the probe Bab di hsp70P. The sequence demonstrated 99-100% and 98% similarity to the 18S rRNA gene fragment of B. divergence and Babesia venatorum, respectively. Molecular biological and serological methods of laboratory diagnosis of babesiosis are considered.


Assuntos
Babesia , Babesiose/diagnóstico , Animais , Técnicas de Laboratório Clínico , Primers do DNA , Diagnóstico Diferencial , Eritrócitos/parasitologia , Humanos , Malária Falciparum , Carrapatos/parasitologia
7.
Plant Dis ; 103(12): 3002-3008, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31573432

RESUMO

Alternaria species are the most important fungal pathogens that attack various crops as well as fruit trees such as pear and cause black spot disease. Here, a loop-mediated isothermal amplification (LAMP) assay is developed for the detection of Alternaria species. A. alternata cytochrome b (cyt-b) gene was used to design two pairs of primers and amplified a 229-bp segment of Aacyt-b gene. The results showed that LAMP assay is faster and simpler than polymerase chain reaction (PCR). LAMP assay is highly sensitive method for the detection of about 1 pg of genomic DNA of A. alternata by using optimized concentration of MgCl2 (4 mM) in final LAMP reaction. In contrast, the limit of detection was 1 ng of target DNA via conventional PCR. Among the genomic DNA of 46 fungal species, only the tubes containing DNA of Alternaria spp. except A. porri, A. solani, and A. infectoria changed color from orange to yellowish green with SYBR Green I including the main pathogens of pear black spot. The yellowish green color was indicative of DNA amplification. Moreover, LAMP assay was used for testing infected tissues among 22 healthy and diseased pear tissues; the orange color changed to yellowish green for infected tissues only. Altogether, we conclude that cyt-b gene can be used for the detection of Alternaria spp. via LAMP assay, which is involved in pear black spot disease.


Assuntos
Alternaria , Técnicas de Amplificação de Ácido Nucleico , Pyrus , Alternaria/genética , Citocromos b/genética , Primers do DNA , Microbiologia de Alimentos/métodos , Limite de Detecção , Reação em Cadeia da Polimerase , Pyrus/microbiologia
8.
Plant Dis ; 103(12): 3251-3258, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31596691

RESUMO

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx). Lxx infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel Lxx-specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published Lxx-specific primers and one universal Leifsonia generic primer pair LayF/LayR. Sugarcane leaf samples were collected from Saccharum spp. hybrids in commercial fields (315 samples) and from germplasm clones of five Saccharum species and Erianthus arundinaceus (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of Lxx genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural Lxx-infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse Leifsonia strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these Leifsonia strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and Lxx-pathogen detection with satisfactory sensitivity and specificity.


Assuntos
Actinomycetales , Reação em Cadeia da Polimerase , Saccharum , Actinomycetales/genética , Primers do DNA/genética , Saccharum/microbiologia , Sensibilidade e Especificidade
9.
Plant Dis ; 103(12): 3265-3273, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31596692

RESUMO

Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (<359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatments showed that autoclaved field soil had significantly reduced DNA amount and quality. The air- or oven-dried soil yielded a lower amount of DNA with similar purity, compared with natural field soil. PCR inhibitors were detected in soil DNA substrates targeting pBluescript II SK(+)-plasmid DNA. Al(NH4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.


Assuntos
Nematoides , Solo , Animais , Primers do DNA/genética , Idaho , Nematoides/genética , North Dakota , Solo/parasitologia
10.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(4): 456-459, 2019 Jul 23.
Artigo em Chinês | MEDLINE | ID: mdl-31612690

RESUMO

OBJECTIVE: To analyze the epidemiological and clinical data of an imported case of visceral leishmaniasis in Henan Province, and explore the method of laboratory diagnosis of kala-azar. METHODS: The epidemiological and clinical data of an imported visceral leishmaniasis patient were analyzed. Leishmania donovani bodies in bone marrow smears were observed microscopically. The antibody was detected by using rK39 dipstick test strips. Two pairs of specific primers, K13A-K13B and LITSR-L5.8S, were used to amplify kinetoplast DNA and internal transcribed spacer of rDNA of the parasite, respectively. RESULTS: The patient had been in the epidemic area of visceral leishmaniasis, and had symptoms such as irregular fever, splenomegaly, pancytopenia, and inversed ratio of albumin and globulin. The amastigotes of L. donovani were found in the bone marrow smears, and rK39 test strip was positive, and the PCR products of K13A-K13B and LITSR-L5.8S were 87 bp and 285 bp respectively. The similarities of the two fragment sequences to the corresponding sequences of L. donovani were 94% and 100%, respectively. CONCLUSIONS: The case is diagnosed as visceral leishmaniasis according to the epidemiological data, clinical manifestations and laboratory test results of the patient, and the pathogen is L. donovani.


Assuntos
Doenças Transmissíveis Importadas , Leishmania donovani , Leishmaniose Visceral , Anticorpos Antiprotozoários/sangue , Medula Óssea/parasitologia , Técnicas de Laboratório Clínico , Doenças Transmissíveis Importadas/diagnóstico , Doenças Transmissíveis Importadas/parasitologia , Primers do DNA , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/diagnóstico , Reação em Cadeia da Polimerase
11.
Fa Yi Xue Za Zhi ; 35(4): 387-392, 2019 Aug.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-31532143

RESUMO

Abstract: Objective Quantitative analysis and comparison of the expression of ribonucleic acid (RNA) from frozen organs and formaldehyde-fixed and paraffin-embedded (FFPE) tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin (ACTB) with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA (5S rRNA) achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs (miRNAs), GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.


Assuntos
Formaldeído , MicroRNAs/análise , Inclusão em Parafina , RNA/análise , Reação em Cadeia da Polimerase em Tempo Real/normas , Primers do DNA , Perfilação da Expressão Gênica , Humanos , Miocárdio
12.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura Ambiente , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
13.
Arch Virol ; 164(11): 2761-2768, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31506786

RESUMO

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per µl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.


Assuntos
Bocavirus/genética , Proteínas do Capsídeo/genética , Vírus da Panleucopenia Felina/genética , Mamastrovirus/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Animais , Bocavirus/isolamento & purificação , Doenças do Gato/diagnóstico , Doenças do Gato/virologia , Gatos , China , Primers do DNA/genética , Fezes/virologia , Vírus da Panleucopenia Felina/isolamento & purificação , Mamastrovirus/isolamento & purificação , Filogenia , Análise de Sequência de DNA
14.
Nat Protoc ; 14(10): 2986-3012, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548639

RESUMO

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Ácidos Nucleicos/análise , Primers do DNA , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Humanos , Leptotrichia/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Engenharia de Proteínas/métodos , RNA Guia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/isolamento & purificação , Ribonucleases/metabolismo , Fluxo de Trabalho , Zika virus/genética , Infecção por Zika virus/sangue , Infecção por Zika virus/urina
15.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 1018-1022, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31484272

RESUMO

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.


Assuntos
Bactérias/genética , DNA Bacteriano/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Replicação de Sequência Autossustentável/métodos , Primers do DNA , Humanos , Sensibilidade e Especificidade
16.
Nucleic Acids Res ; 47(16): 8708-8719, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392993

RESUMO

Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The second half of the integration reaction remains very poorly understood. Second-strand DNA cleavage and second-strand DNA synthesis were investigated in vitro using purified components from a site-specific restriction-like endonuclease (RLE) bearing LINE. DNA structure was shown to be a critical component of second-strand DNA cleavage. A hitherto unknown and unexplored integration intermediate, an open '4-way' DNA junction, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the 4-way junction resulted in a natural primer-template pairing used for second-strand DNA synthesis. A new model for RLE LINE integration is presented.


Assuntos
Enzimas de Restrição do DNA/genética , DNA Cruciforme/genética , Elementos Nucleotídeos Longos e Dispersos , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , Transcrição Reversa , Animais , Bombyx/genética , Bombyx/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Clivagem do DNA , Primers do DNA/genética , Primers do DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Cruciforme/química , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo
17.
Fitoterapia ; 138: 104295, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400481

RESUMO

Gynostemma pentaphyllum is a traditional oriental medicinal herb used as tea since ancient time. Among Gynostemma species, G. pentaphyllum has more active chemical components and better therapeutic effect. It is used to cure depression, diabetes, anxiety, hyperlipidemia, fatigue, immunity, cancer, and oxidative stress. Overexploitation of G. pentaphyllum for its medicinal benefits has been on a rise, due to which they are adulterated or mistakenly identified with other members of Gynostemma species. Hence, we used chloroplast universal regions such as ycf3, accD, petD, psbB and their polymorphism to distinguish G. pentaphyllum from other Gynostemma species. By using the species-specific primers derived from the above regions, we established a multiplex allele-specific PCR for the authentication of G. pentaphyllum from other species. Thus the PCR reaction produced unique amplicons of size 244 bp and 438 bp for G. pentaphyllum amplified by the primers flanking ycf3, and accD regions respectively. While a 607 bp, and 787 bp amplicons from the primers targeting psbB, and petD regions distinguished G. longipes, G. burmanicum, and G. pubescens species. Moreover, these primers were successful to analyze the dried tea samples of Gynostemma as well. Thus, the developed molecular markers could authenticate different Gynostemma species as well as its products thereby preventing the mistaken-identity of this medicinal herb.


Assuntos
Primers do DNA/genética , Genes de Cloroplastos , Gynostemma/classificação , Plantas Medicinais/classificação , Sequência de Bases , Biomarcadores , Cloroplastos , Genes de Plantas , Filogenia , Controle de Qualidade , República da Coreia , Especificidade da Espécie
18.
Mar Pollut Bull ; 146: 827-830, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31426224

RESUMO

The invasive Crepidula fornicata caused major problems along the European Atlantic coast, especially in France and Netherlands where high densities leads on changes in the habitat, disturb native marine wildlife as well as it originates competition for space and food. Despite its dangerous invasive nature, regular monitoring to alert about its presence in risk areas, like the south Bay of Biscay (Spain and south France), is not done yet. Here, we developed a species-specific marker to detect the presence of C. fornicata in environmental samples (eDNA) of seawater. The novel C. fornicata specific primers amplified a region of 239 bp within the COI gen. We employed this tool to check its presence in 6 estuaries of the Cantabrian Sea, an area comprised between the Spanish and French limits of the previously reported presence of this limpet in the south Bay of Biscay. The presence of C. fornicata was confirmed in A Coruña (Galicia, Spain), Eo and Villaviciosa estuaries (Asturias, Spain) while it was not detected in Santander, Bilbao (Spain), and Bayonne (France). This new method to detect C. fornicata could be easily implemented in regular monitoring to prevent and manage future invasions of this species.


Assuntos
Monitoramento Ambiental/métodos , Gastrópodes/genética , Marcadores Genéticos , Reação em Cadeia da Polimerase/métodos , Animais , Primers do DNA , Ecossistema , França , Espécies Introduzidas , Países Baixos , Água do Mar , Espanha , Especificidade da Espécie
19.
Plant Dis ; 103(11): 2893-2902, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31436473

RESUMO

Uniqprimer, a software pipeline developed in Python, was deployed as a user-friendly internet tool in Rice Galaxy for comparative genome analyses to design primer sets for PCRassays capable of detecting target bacterial taxa. The pipeline was trialed with Dickeya dianthicola, a destructive broad-host-range bacterial pathogen found in most potato-growing regions. Dickeya is a highly variable genus, and some primers available to detect this genus and species exhibit common diagnostic failures. Upon uploading a selection of target and nontarget genomes, six primer sets were rapidly identified with Uniqprimer, of which two were specific and sensitive when tested with D. dianthicola. The remaining four amplified a minority of the nontarget strains tested. The two promising candidate primer sets were trialed with DNA isolated from 116 field samples from across the United States that were previously submitted for testing. D. dianthicola was detected in 41 samples, demonstrating the applicability of our detection primers and suggesting widespread occurrence of D. dianthicola in North America.


Assuntos
Agricultura , Técnicas Bacteriológicas , Primers do DNA , Enterobacteriaceae , Solanum tuberosum , Agricultura/métodos , Técnicas Bacteriológicas/métodos , Primers do DNA/genética , Enterobacteriaceae/genética , América do Norte , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia
20.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1463-1468, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441617

RESUMO

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.


Assuntos
Produtos do Gene tat , Membrana Celular , Primers do DNA , Escherichia coli , Expressão Gênica , Vetores Genéticos , Proteínas Recombinantes de Fusão
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