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1.
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
2.
Parasit Vectors ; 12(1): 360, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340841

RESUMO

BACKGROUND: Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37-42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1-10 target DNA copies in less than 20 minutes. METHODS: We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye. RESULTS: The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20-25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field. CONCLUSIONS: Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.


Assuntos
Alveolados/isolamento & purificação , Crassostrea/parasitologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Protozoárias em Animais/diagnóstico , Alveolados/genética , Animais , Primers do DNA/genética , DNA Intergênico/genética , Visualização de Dados , Eletroforese em Gel de Ágar , Reação em Cadeia da Polimerase/métodos , Infecções por Protozoários , Recombinases/genética , Sensibilidade e Especificidade
3.
J Med Microbiol ; 68(9): 1324-1329, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31355739

RESUMO

Purpose. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK).Methodology. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK.Results. There was no difference in the performance of both primer sets for all HSV-1 dilutions (P=0.83) using a CIM or between a CIM and a swab (P=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (P=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (P=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (P<0.001).Conclusions. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.


Assuntos
Córnea/virologia , Herpes Simples/diagnóstico , Herpesvirus Humano 1/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Primers do DNA/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
Arch Virol ; 164(10): 2581-2584, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31359148

RESUMO

Maize chlorotic mottle virus (MCMV), an important quarantine virus, causes lethal necrosis in maize when coinfected with a potyvirid, which is seriously threatening the production of maize worldwide. In this study, recombinase polymerase amplification (RPA), a novel isothermal DNA amplification and detection technique, was developed to detect MCMV in maize crops. A pair of specific primers was designed based on the conserved sequences of the MCMV coat protein region. The RT-RPA assay was carried out as an isothermal reaction at 38 °C that was complete within 30 min, and no cross-reactivity was detected with other viruses infecting maize in China. The limit of detection of the RT-RPA assay was tenfold lower than that of ordinary RT-PCR. Moreover, this method was successfully applied to test field-collected samples. The newly developed RT-RPA assay offers a reliable, sensitive and efficient method for rapid detection of MCMV in maize in equipment-limited diagnostic laboratories and on-site facilities.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Tombusviridae/isolamento & purificação , Proteínas do Capsídeo/genética , China , Primers do DNA/genética , Sensibilidade e Especificidade , Temperatura Ambiente , Fatores de Tempo , Tombusviridae/classificação , Tombusviridae/genética
5.
World J Microbiol Biotechnol ; 35(6): 95, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187258

RESUMO

Recombinase polymerase amplification (RPA) is an isothermal amplification technique. Because of its short detection cycle and high specificity, it has been applied in various fields. However, the design of probe on the efficiency of RPA is not well understood and the effect of sequence mismatches of oligonucleotides on the performance of RPA is rarely discussed. In this study, we found that different primers with the same probe have a slight effect on the efficiency of fluorescent RPA, and different probes with the same amplified region have a great influence on the efficiency of fluorescent RPA. We summarized the design rules of probes suitable for fluorescent RPA by analyzing the experimental data. The rule is that the best distance between fluorescent groups in the probe is 1-2 bases, and the G content should be reduced as far as possible. In addition, we verified this rule by designing a series of probes. Furthermore, we found the base mismatches of the probe had a significant effect on RPA, which can lead to false positives and can change the amplification efficiency. However, 1-3 mismatches covering the center of the primer sequence only affect the amplification efficiency of RPA, not its specificity. And with an increase in the number of primer mismatches, the efficiency of RPA will decrease accordingly. This study suggests that the efficiency of fluorescent RPA is closely related to the probe. We recommend that when designing a fluorescent probe, one must consider the presence of closely related non-targets and specific bases.


Assuntos
Pareamento Incorreto de Bases , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases , Bactérias , Primers do DNA/genética , Sensibilidade e Especificidade
6.
Zhonghua Nei Ke Za Zhi ; 58(6): 419-422, 2019 Jun 01.
Artigo em Chinês | MEDLINE | ID: mdl-31159519

RESUMO

Objective: To study the significance of Th17 cells in patients with myelodysplastic syndrome (MDS) and iron overload. Methods: A total of 77 patients with MDS admitted to Guangzhou First People's Hospital were enrolled from January 2017 to December 2018,who were divided into iron overload group (37 cases) with serum ferritin (SF) over 1000 µg/L and non-ferrous overload group(40 cases). CD(4)(+)T cells in peripheral blood (PB) and bone marrow (BM) were sorted by flow cytometry. The ratio of Th17 cells and cells with abnormal karyotype were compared. IL-17 and IL-6 protein and RNA expression were detected by ELISA and quantitative real-time PCR(qRT-PCR). Results: The proportions of Th17 cells in PB and BM in iron overload group were significantly higher than those in non-iron overload group [(41.06±0.96)% vs. (26.80±1.21)%; (47.39±1.60)% vs. (34.29±1.03)%; P<0.01]. The Th17 positive cells with abnormal karyotype in iron overload group were more than those in non-iron overload group[(4.96±0.53)% vs. (3.67±0.12)% in PB; (10.06±1.67)% vs. (4.36±0.43)% in BM; P<0.01]. Similarly,the protein levels as well as mRNA expression of IL-6 and IL-17 in patients with iron overload were significantly higher than those in non-iron overload group (P<0.01 both in PB and BM). Conclusions: As hematopoietic regulators secreted by Th17 cells, the expression of IL-6 and IL-17 in MDS patients with iron overload are elevated. This may predict the influence of these factors to the differentiation of Th17 cells.


Assuntos
Interleucina-17/imunologia , Interleucina-6/imunologia , Interleucinas/imunologia , Sobrecarga de Ferro , Síndromes Mielodisplásicas/sangue , Células Th17/imunologia , Medula Óssea , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Ferritinas/sangue , Citometria de Fluxo , Humanos , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Interleucinas/metabolismo , Ferro/sangue , Síndromes Mielodisplásicas/imunologia , RNA , Reação em Cadeia da Polimerase em Tempo Real
7.
Parasit Vectors ; 12(1): 312, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234937

RESUMO

BACKGROUND: Babesiosis is an economically important disease caused by tick-borne apicomplexan protists of the genus Babesia. Most apicomplexan parasites, including Babesia, have a plastid-derived organelle termed an apicoplast, which is involved in critical metabolic pathways such as fatty acid, iron-sulphur, haem and isoprenoid biosynthesis. Apicoplast genomic data can provide significant information for understanding and exploring the biological features, taxonomic and evolutionary relationships of apicomplexan parasites, and identify targets for anti-parasitic drugs. However, there are limited data on the apicoplast genomes of Babesia species infective to small ruminants. METHODS: PCR primers were designed based on the previously reported apicoplast genome sequences of Babesia motasi Lintan and Babesia sp. Xinjiang using Illumina technology. The overlapped apicoplast genomic fragments of six ovine Babesia isolates were amplified and sequenced using the Sanger dideoxy chain-termination method. The full-length sequences of the apicoplast genomes were assembled and annotated using bioinformatics software. The gene contents and order of apicoplast genomes obtained in this study were defined and compared with those of other apicomplexan parasites. Phylogenetic trees were constructed on the concatenated amino acid sequences of 13 gene products using MEGA v.6.06. RESULTS: The results showed that the six ovine Babesia apicoplast genomes consisted of circular DNA. The genome sizes were 29,916-30,846 bp with 78.7-81.0% A + T content, 29-31 open reading frames (ORF) and 23-24 transport RNAs. The ORFs encoded four DNA-directed RNA polymerase subunits (rpoB, rpoCl, rpoC2a and rpoC2b), 13 ribosomal proteins, one elongation factor TU (tufA), two ATP-dependent Clp proteases (ClpC) and 7-11 hypothetical proteins. Babesia sp. has three more genes than Babesia motasi (rpl5, rps8 and rpoB). Phylogenetic analysis showed that Babesia sp. is located in a separate clade. Babesia motasi Lintan/Tianzhu and B. motasi Ningxian/Hebei were divided into two subclades. CONCLUSIONS: To our knowledge, this study is the first to elucidate the whole apicoplast genomic structural features of six Babesia isolates infective to small ruminants in China using Sanger sequencing. The data provide useful information confirming the taxonomic relationships of these parasites and identifying targets for anti-apicomplexan parasite drugs.


Assuntos
Apicoplastos/genética , Babesia/genética , Genoma de Protozoário , Ruminantes/parasitologia , Animais , Babesiose/epidemiologia , China , Biologia Computacional , Primers do DNA/genética , Anotação de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/parasitologia
8.
Vet Parasitol ; 269: 2-6, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31079823

RESUMO

Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira, were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle.


Assuntos
Aborto Animal/diagnóstico , Doenças dos Bovinos/diagnóstico , Coccidiose/veterinária , Neospora/isolamento & purificação , Complicações Parasitárias na Gravidez/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Feto Abortado/parasitologia , Aborto Animal/parasitologia , Animais , Encéfalo/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/diagnóstico , Coccidiose/parasitologia , Primers do DNA/genética , Sondas de DNA/genética , Feminino , Coração/parasitologia , Neospora/genética , Gravidez , Complicações Parasitárias na Gravidez/diagnóstico , Complicações Parasitárias na Gravidez/parasitologia
9.
J Microbiol Biotechnol ; 29(6): 984-988, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31091865

RESUMO

Blue mold in citrus is caused by Penicillium italicum. In this study, the P. italicum-specific primers were developed for rapid detection based on the conserved genes RPB1 and RPB2 among Penicillium genomes. The two primer pairs RPB1-a and RPB1-b proved to be specific to detect P. italicum. The PCR assay among 39 fungal isolates and the colonial, pathogenic morphologies and molecular methods validated the specificity and reliability of these two primer pairs. This report provided a method and P. italicum-specific primers, which might greatly contribute to citrus postharvest industry.


Assuntos
Citrus/microbiologia , Primers do DNA/normas , Microbiologia de Alimentos/métodos , Técnicas de Tipagem Micológica/métodos , Penicillium/genética , Doenças das Plantas/microbiologia , Proteínas de Bactérias/genética , Primers do DNA/genética , Penicillium/classificação , Reação em Cadeia da Polimerase , RNA Polimerase II/genética , Reprodutibilidade dos Testes , Especificidade da Espécie
10.
Arch Virol ; 164(6): 1691-1695, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30968213

RESUMO

Potato virus Y (PVY) is the most common virus infecting potato worldwide. We analysed potato tuber PVY infections from the major Israeli growing region in 2014-2017. Isolates were characterized by multiplex PCR according to Chikh-Ali et al. (Plant Disease 97, 1370, 2013), whose primers were not fully compatible with the Israeli isolates. New primers were designed for a multiplex PCR assay to differentiate the Israeli isolates. Three recombinant strains were observed: PVYNTNa (72% of the isolates), PVYNWi (24%) and PVYSyr-III (found only in 2015). The archetypal PVYO strain was found only once. The classical PVY strains have recently been displaced by recombinant forms, with PVYNTNa dominating. The Israeli isolates appear very similar to those of Europe (the seed tuber source), except for PVYSyr-III.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Potyvirus/isolamento & purificação , Solanum tuberosum/virologia , Primers do DNA/genética , Genoma Viral , Israel , Doenças das Plantas/virologia , Potyvirus/genética , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Análise de Sequência de RNA
11.
Malar J ; 18(1): 116, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940128

RESUMO

BACKGROUND: Malaria remains a global public health problem responsible for 445,000 deaths in 2016. While microscopy remains the mainstay of malaria diagnosis, highly sensitive molecular methods for detection of low-grade sub-microscopic infections are needed for surveillance studies and identifying asymptomatic reservoirs of malaria transmission. METHODS: The Plasmodium falciparum genome sequence was analysed to identify high copy number genes that improve P. falciparum parasite detection in blood by RT-PCR. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)-specific primers were evaluated for P. falciparum detection in hospital-based microscopically positive dried blood spots and field-acquired whole blood from asymptomatic individuals from Ghana. RESULTS: PfEMP1 outperformed the Pf18S sequence for amplification-based P. falciparum detection. PfEMP1 primers exhibited sevenfold higher sensitivity compared to Pf18S primers for parasite genomic DNA. Probit analysis established a 95% detection threshold of 9.3 parasites/mL for PfEMP1 compared to 98.2 parasites/mL for Pf18S primers. The PfEMP1 primers also demonstrated superior clinical sensitivity, identifying 100% (20/20) of dried blood spot samples and 70% (69/98) of asymptomatic individuals as positive versus 55% (11/20) and 54% (53/98), respectively, for Pf18S amplification. CONCLUSIONS: These results establish PfEMP1 as a novel amplification target for highly sensitive detection of both acute infections from filter paper samples and submicroscopic asymptomatic low-grade infections.


Assuntos
Sangue/parasitologia , Malária Falciparum/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Doenças Assintomáticas , Criança , Pré-Escolar , Primers do DNA/genética , Feminino , Gana , Humanos , Lactente , Masculino , Programas de Rastreamento/métodos , Plasmodium falciparum/genética , Sensibilidade e Especificidade
12.
Methods Mol Biol ; 1979: 197-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028640

RESUMO

Single-cell sequencing of TCR alleles enables determination of T cell specificity. Here we describe a sensitive protocol for targeted amplification of TCR CDR3 regions from single-cell full-length cDNA libraries. By exploiting the specificity of RNase H-dependent PCR (rhPCR), the protocol achieves amplification of TCR alleles and addition of cell barcodes in a single PCR step.


Assuntos
Alelos , Biblioteca Gênica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Célula Única/métodos , Sequência de Bases , Regiões Determinantes de Complementaridade/genética , Primers do DNA/genética , Humanos , Reação em Cadeia da Polimerase/métodos
14.
Clin Lab ; 65(4)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969069

RESUMO

BACKGROUND: Escherichia coli is the most common pathogenic bacteria that frequently causes life-threatening opportunistic human infections, diarrhea, and septicemia in immunocompromised hosts. METHODS: This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of a hypothetical protein from an E. coli-specific gene (GenBank ID: 13702648). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65ºC for 30 minutes and 80ºC for 2 minutes, whereas the reaction system contained 5.2 mM Mg2+, 8 U of Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide, and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 240 strains of E. coli and 150 strains of non-E. coli. RESULTS: Positive reactions were observed on all 240 strains of E. coli while all non-E. coli strains were negative. Plasmids with the specific gene and mice blood with E. coli were used for sensitivity analysis. The detection limit of LAMP was 100 bacterium/reaction. CONCLUSIONS: Results showed that the LAMP targeted to the hypothetical protein (GenBank ID: 13702648) is a fast, specific, sensitive, inexpensive, and suitable method for the detection of E. coli.


Assuntos
Técnicas de Laboratório Clínico/métodos , Escherichia coli/genética , Técnicas de Amplificação de Ácido Nucleico , Animais , Primers do DNA/genética , Diarreia/microbiologia , Humanos , Hospedeiro Imunocomprometido , Limite de Detecção , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
15.
Clin Lab ; 65(4)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969072

RESUMO

BACKGROUND: Schizophrenia (SCZ) is a serious mental disorder that interferes with a person's cognitive processes and leads to social disability. A wide range of factors may play important roles in increased risk of SCZ development. Genetic contributors are among the most influential actors involved in different molecular mechanisms leading to the development of the nervous system, thus they play pivotal roles in psychotic disorders and SCZ de-velopment. RAB8B is characterized for its key roles in several cellular and molecular mechanisms which are linked with different psychotic disorders, such as SCZ. METHODS: In this study, we assessed the expression level of RAB8B gene in blood samples of schizophrenic patients and normal healthy controls by means of quantitative real time PCR. We also investigated the correlation between RAB8B-rs1986112 genotypes and RAB8B expression levels through SNP genotyping by means of the PCR-RFLP method. RESULTS: Our results indicated a significant difference of RAB8B mRNA ratio between SCZ patients and healthy controls. Moreover, we showed significant upregulation of RAB8B in patients with rs1986112 GG and AG genotype compared to AA genotype. CONCLUSIONS: Our findings suggest a role for RAB8B and its regulatory variation, rs1986112 in SCZ development.


Assuntos
Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Polimorfismo de Nucleotídeo Único , Esquizofrenia/genética , Adulto , Estudos de Casos e Controles , Cognição , Biologia Computacional , Primers do DNA/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo Real , Risco , Regulação para Cima
16.
Appl Microbiol Biotechnol ; 103(12): 4943-4952, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025076

RESUMO

Swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. To develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (TGEV) N, porcine epidemic diarrhea virus (PEDV) M, porcine deltacoronavirus (PDCoV) M, and porcine enteric alphacoronavirus (PEAV) N genes respectively. A TaqMan-probe-based multiplex real-time RT-qPCR assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. The results showed that the limit of detection can reach as low as 10 copies in singular real-time RT-qPCR assays and 100 copies in multiplex real-time RT-qPCR assay, with all correlation coefficients (R2) at above 0.99, and the amplification efficiency at between 90 and 120%. This multiplex real-time RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. The multiplex real-time RT-qPCR assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. The results manifested that TGEV and PDCoV were the main pathogens in these samples, accompanied by co-infections. This well-established multiplex real-time RT-qPCR assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Doenças dos Suínos/diagnóstico , Animais , Coronavirus/classificação , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Diagnóstico Diferencial , Diarreia/virologia , Fezes/virologia , Gastroenterite Suína Transmissível/diagnóstico , Gastroenterite Suína Transmissível/virologia , Limite de Detecção , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/virologia , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/isolamento & purificação
17.
J Pharm Biomed Anal ; 170: 196-203, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30928895

RESUMO

In this study, probe/primers of high specificity and sensitivity were selected to analyze donkey-hide gelatin for donkey DNA and to look for horse, ox, and pig DNA as possible adulterants. The mitochondrial CO I genes in donkey, horse, and ox were selected as target sequences for design and synthesis of three pairs of specific probes and primers. In addition, eight pairs of probe/primers were obtained via literature search. Out of these eleven groups of probe/primers, those with the highest specificity and sensitivity were selected, which was fulfilled by the screening firstly with animal hide samples then the hide-glue samples. Other parameters that might affect detection specificity and efficiency-such as the amount of sampling and final concentration of primers-were also optimized. Replication tests were also conducted. The results showed that the selected probe/primers could accurately detect donkey DNA and horse, ox, and pig DNA in gelatin samples with good reproducibility. Analysis of four samples of on-market gelatin using this assay showed that two of the four samples indeed contained only donkey DNA, whereas the other two samples contained both donkey and horse DNA, indicating adulteration of these samples with horse hide. These results indicate that the TaqMan probe real-time PCR method can be used for identifying the purity of donkey DNA in gelatin samples, and can provide technical support for identifying adulterations in the gelatin market.


Assuntos
Equidae/genética , Gelatina/genética , Animais , DNA/genética , Primers do DNA/genética , Cavalos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos
18.
Lett Appl Microbiol ; 69(1): 64-70, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31021437

RESUMO

Phytophthora capsici is an important oomycete pathogen that causes devastating diseases in various crops. Methods for the rapid detection of P. capsici are important for disease control and eradication programmes. Here, we developed an assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of P. capsici. The specific primers and a probe were designed using the sequence of Ypt1, and the optimal assay conditions were 40°C for 20 min. The specificity of the assay was verified using closely related oomycetes and fungal species, and its detection limit was 10 pg of genomic DNA. In combination with a simple DNA extraction method, the LF-RPA assay enabled detection of P. capsici in diseased pepper samples without specialized equipment within 30 min. Consequently, the LF-RPA assay developed in this study enables rapid and equipment-free detection of P. capsici and has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a novel assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of Phytophthora capsici. In combination with a simple DNA extraction method, the LF-RPA assay detected P. capsici in field samples without specialized equipment within 30 min. The assay has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Phytophthora/genética , Phytophthora/isolamento & purificação , Doenças das Plantas/parasitologia , Recombinases/análise , Alquil e Aril Transferases/genética , Primers do DNA/genética , Limite de Detecção , Sensibilidade e Especificidade
19.
J Clin Pathol ; 72(7): 487-492, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30952829

RESUMO

AIMS: Helminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay. METHODS: We designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested. RESULTS: The PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples. CONCLUSIONS: The highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.


Assuntos
Cestoides/isolamento & purificação , Infecções por Cestoides/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Nematoides/isolamento & purificação , Infecções por Nematoides/diagnóstico , Trematódeos/isolamento & purificação , Infecções por Trematódeos/diagnóstico , Animais , Sequência de Bases , Cestoides/genética , Infecções por Cestoides/parasitologia , Estudos de Coortes , Primers do DNA/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Fezes/parasitologia , Humanos , Nematoides/genética , Infecções por Nematoides/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Alinhamento de Sequência , Trematódeos/genética , Infecções por Trematódeos/parasitologia
20.
Virus Res ; 265: 162-165, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30930200

RESUMO

The complete sequence was obtained for two variants of raspberry vein chlorosis virus (RVCV), confirming that this virus is a rhabdovirus most closely related to the cytorhabdoviruses alfalfa dwarf virus and strawberry crinkle virus. The two RVCV variants share only a 68% nucleotide sequence identity so that the previously published RT-PCR diagnostic test for this virus was not able to efficiently detect both variants. Using the new, complete sequence information several new primer sets have been designed that allow a much improved RVCV detection.


Assuntos
Primers do DNA/genética , Genoma Viral , Vírus de Plantas/genética , Rhabdoviridae/genética , Rubus/virologia , Variação Genética , Filogenia , Doenças das Plantas/virologia , Reação em Cadeia da Polimerase/métodos , RNA Viral , Análise de Sequência de DNA
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