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Article in English | WPRIM | ID: wpr-939589


Objective@#To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .@*Methods@#We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).@*Results@#CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g.@*Conclusion@#The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

CRISPR-Cas Systems , Nucleic Acid Amplification Techniques/methods , Recombinases/genetics , Vibrio parahaemolyticus/genetics
Article in English | WPRIM | ID: wpr-927643


OBJECTIVE@#To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene.@*METHODS@#A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay.@*RESULTS@#The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses.@*CONCLUSION@#A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

African Swine Fever/virology , African Swine Fever Virus/isolation & purification , Animals , Nucleic Acid Amplification Techniques/methods , Recombinases/chemistry , Sensitivity and Specificity , Swine , Viral Proteins/genetics
Article in English | WPRIM | ID: wpr-887737


Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Antitubercular Agents , Bacterial Proteins/genetics , Catalase/genetics , DNA, Bacterial/analysis , DNA-Directed RNA Polymerases/genetics , Drug Resistance, Multiple, Bacterial/genetics , Isoniazid , Molecular Diagnostic Techniques/methods , Mutation , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Amplification Techniques/methods , Oxidoreductases/genetics , Phenotype , Rifampin , Whole Genome Sequencing
Rev. cuba. med. trop ; 72(2): e522, mayo.-ago. 2020. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1149912


Introducción: Los ensayos para cuantificar el ADN del virus de la hepatitis B (VHB) o carga viral son imprescindibles en el diagnóstico y en el seguimiento de los pacientes con hepatitis B crónica; de ahí que estén disponibles estuches diagnósticos para esta función. En el presente estudio se muestra la validación de SUMASIGNAL VHB (un paso), el cual es un sistema de reacción en cadena de la polimerasa en tiempo real (RCP-TR) para la cuantificación del genoma del VHB, propuesto por el Centro de Inmunoensayo. Objetivo: Evaluar el desempeño analítico de SUMASIGNAL VHB (un paso). Métodos: Se utilizó un panel de 80 muestras de suero bien caracterizadas y el Tercer Estándar Internacional de la OMS para las técnicas de amplificación de ácidos nucleicos del virus de la hepatitis B. Se determinaron las características del ensayo como especificidad clínica, especificidad analítica (reactividad cruzada), rango lineal o linealidad y exactitud, precisión intraensayo y comparación con un ensayo de referencia. Resultados: La especificidad analítica y clínica fue del 100 por ciento. Al evaluar la linealidad y exactitud con un estándar de referencia de la OMS, se obtuvo que la totalidad de las diferencias entre los Log10 del valor obtenido y el de referencia resultaron inferiores a 0,5 Log10 (r= 0,9977 y r2= 0,9954). Además, se obtuvieron bajos coeficientes de variación intraensayo. La evaluación comparativa con el estuche comercial Artus HBV RG PCR kit mostró una correlación fuerte (r= 0,8882). Conclusiones: SUMASIGNAL VHB (un paso) es un ensayo fácil de realizar manualmente, es rápido e incluye reactivos de extracción de ácidos nucleicos. Teniendo en cuenta la validez del método para el uso previsto, puede ser recomendado para su introducción en el diagnóstico, la vigilancia y la indicación de tratamiento en los pacientes con hepatitis B crónica(AU)

Introduction: Assays to quantify hepatitis B virus (HBV) DNA or viral load are indispensable for the diagnosis and follow-up of patients with chronic hepatitis B, hence the availability of diagnostic kits for this purpose. The present study deals with the validation of HBV SUMASIGNAL (one step), a real time polymerase chain reaction (RT-PCR) system for quantification of the HBV genome proposed by the Immunoassay Center. Objective: Evaluate the analytical performance of HBV SUMASIGNAL (one step). Methods: Use was made of a panel of 80 well characterized serum samples and the Third WHO International Standard for hepatitis B virus nucleic acid amplification techniques. Determination was performed of assay characteristics such as clinical specificity, analytical specificity (cross-reactivity), linear range or linearity and accuracy, intra-assay precision and comparison with a reference assay. Results: Analytical and clinical specificity was 100 percent. Evaluation of linearity and accuracy with a WHO reference standard revealed that all the differences between the log10 of the value obtained and the reference value were lower than 0.5 log10 (r= 0.9977 and r2= 0.9954). The intra-assay variation coefficients obtained were low. Comparative evaluation with the commercial Artus HBV RG PCR kit showed a strong correlation (r= 0.8882). Conclusions: The assay HBV SUMASIGNAL (one step) is easy to conduct manually, fast and includes reagents for nucleic acid extraction. Based on the validity of the method for the use in mind, it may be recommended for incorporation into the diagnosis, surveillance and treatment of patients with chronic hepatitis B(AU)

Humans , Hepatitis B virus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Real-Time Polymerase Chain Reaction/methods , Validation Study
Mem. Inst. Oswaldo Cruz ; 115: e200006, 2020. tab, graf
Article in English | LILACS, SES-SP | ID: biblio-1135222


BACKGROUND Occult hepatitis B virus (HBV) - characterized by the absence of detectable HBsAg in the presence of HBV DNA - represents a potential threat for blood safety. OBJECTIVES This study was conducted with the aim to investigate the serological and molecular characterization of occult HBV infection (OBI) among blood donors in Mozambique. METHODS 1,502 blood donors were tested for HBsAg. All HBsAg-negative individuals were tested for HBV DNA. Antibodies against HBV core, surface and HBe antigen (anti-HBc, anti-HBs, HBeAg) were measured in HBV DNA positive individuals. FINDINGS 1435 serum samples were HBsAg negative and 16 positive for HBV DNA, 14 confirmed to have OBI, corresponding to a frequency of 0.98%. Of the 14 OBI infections identified, 13/14 (92.8%) were positive for anti-HBc, 4/14 (28.5%) for anti-HBs, and no samples were reactive for HBeAg. Of the 14 OBI cases, nine samples (64.2%) were sequenced for the S/P region. Eight samples (88.9%) belonged to genotype A1 and one (11.1%) to genotype E. One escape mutation (T123A) associated with OBI and various amino acid substitutions for genotype A1 and E were observed. MAIN CONCLUSIONS Our results show the importance of using nucleic acid amplification test to detect occult hepatitis B infection in blood donors in Mozambique.

Humans , Male , Female , Adult , Blood Donors , Hepatitis B virus/isolation & purification , Hepatitis B virus/genetics , Nucleic Acid Amplification Techniques/methods , Hepatitis B/diagnosis , Hepatitis B Surface Antigens/genetics , Phylogeny , DNA, Viral , Polymerase Chain Reaction , Cross-Sectional Studies , Mozambique
Article in Chinese | WPRIM | ID: wpr-879921


OBJECTIVE@#To evaluate the effect of standardized health education on the sputum specimen collection rate for nucleic acid detection of coronavirus disease 2019 (COVID-19).@*METHODS@#Two hundred and twenty-seven patients in fever clinics and isolation wards of Sir Run Run Shaw Hospital of Zhejiang University and 307 migrant workers returning to 5 enterprises in Shanghai from February 3 to March 14, 2020 were enrolled in the study. Through clarifying the procedures of collecting sputum specimens, making graphic/video health education materials, standardizing the contents and methods of health education, we conducted education to the subjects. The subject expectorated spontaneously or with medical assistance. For patients, the number of sampling attempts and sputum acquisition times were documented before and after the implementation of the standardized expectoration method; for the returning migrant employees in the enterprises, only the number of collected samples after the implementation of the standardized expectoration method were recorded.@*RESULTS@#A total of 378 sputum samples were collected from 227 patients. The sputum sampling rates before and after the implementation of health education were 40.9%and 58.4%, respectively (@*CONCLUSIONS@#The education for standardized sputum sample collection method can effectively increase the sputum collection rate.

Betacoronavirus/genetics , COVID-19 , China , Coronavirus Infections/diagnosis , Efficiency , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , SARS-CoV-2 , Specimen Handling/methods , Sputum , Time Factors
J. bras. pneumol ; 45(2): e20170451, 2019. tab
Article in English | LILACS | ID: biblio-1040271


ABSTRACT Tuberculosis continues to be a major public health problem worldwide. The aim of the present study was to evaluate the accuracy of the Xpert MTB/RIF rapid molecular test for tuberculosis, using pulmonary samples obtained from patients treated at the Júlia Kubitschek Hospital, which is operated by the Hospital Foundation of the State of Minas Gerais, in the city of Belo Horizonte, Brazil. This was a retrospective study comparing the Xpert MTB/RIF test results with those of standard culture for Mycobacterium tuberculosis and phenotypic susceptibility tests. Although the Xpert MTB/RIF test showed high accuracy for the detection of M. tuberculosis and its resistance to rifampin, attention must be given to the clinical status of the patient, in relation to the test results, as well as to the limitations of molecular tests.

RESUMO A tuberculose permanece como um grave problema de saúde pública. O objetivo deste estudo foi avaliar a acurácia do teste rápido molecular Xpert MTB/RIF em amostras pulmonares no Hospital Júlia Kubitschek, Fundação Hospitalar do Estado de Minas Gerais, localizado em Belo Horizonte (MG). Trata-se de um estudo descritivo retrospectivo, considerando-se como método padrão a cultura para o bacilo da tuberculose e o teste de sensibilidade fenotípico. O teste Xpert MTB/RIF apresentou ótima acurácia para a detecção da tuberculose e resistência à rifampicina, mas é necessária a atenção a dados clínicos do paciente em relação ao resultado do exame e às limitações dos testes moleculares.

Humans , Sputum/microbiology , Trachea/microbiology , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/microbiology , Bronchoalveolar Lavage Fluid/microbiology , Nucleic Acid Amplification Techniques/methods , Rifampin/pharmacology , DNA, Bacterial , Reproducibility of Results , Retrospective Studies , Sensitivity and Specificity , Molecular Diagnostic Techniques/methods , Drug Resistance, Bacterial/drug effects , Tertiary Care Centers , Antibiotics, Antitubercular/pharmacology , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/drug effects
J. bras. pneumol ; 45(2): e20180128, 2019. tab, graf
Article in English | LILACS | ID: biblio-1002440


ABSTRACT Objective: To evaluate the rapid diagnosis of multidrug-resistant tuberculosis, by using a commercial line probe assay for rifampicin and isoniazid detection (LPA-plus), in the routine workflow of a tuberculosis reference laboratory. Methods: The LPA-plus was prospectively evaluated on 341 isolates concurrently submitted to the automated liquid drug susceptibility testing system. Results: Among 303 phenotypically valid results, none was genotypically rifampicin false-susceptible (13/13; 100% sensitivity). Two rifampicin-susceptible isolates harboured rpoB mutations (288/290; 99.3% specificity) which, however, were non-resistance-conferring mutations. LPA-plus missed three isoniazid-resistant isolates (23/26; 88.5% sensitivity) and detected all isoniazid-susceptible isolates (277/277; 100% specificity). Among the 38 (11%) invalid phenotypic results, LPA-plus identified 31 rifampicin- and isoniazid-susceptible isolates, one isoniazid-resistant and six as non-Mycobacterium tuberculosis complex. Conclusions: LPA-plus showed excellent agreement (≥91%) and accuracy (≥99%). Implementing LPA-plus in our setting can speed up the diagnosis of multidrug-resistant tuberculosis, yield a significantly higher number of valid results than phenotypic drug susceptibility testing and provide further information on the drug-resistance level.

RESUMO Objetivo: Avaliar o diagnóstico rápido de tuberculose multirresistente, utilizando um teste comercial de sondas em linha (LPA-plus), na rotina de um laboratório de referência de tuberculose. Métodos: O teste LPA-plus foi avaliado prospectivamente em 341 isolados simultaneamente submetidos ao teste de suscetibilidade aos antimicrobianos em meio líquido, pelo sistema automatizado. Resultados: Entre os 303 resultados fenotipicamente válidos, nenhum foi genotipicamente falso suscetível à rifampicina (13/13; 100% de sensibilidade). Dois isolados sensíveis à rifampicina apresentavam mutações no gene rpoB (288/290; especificidade de 99,3%), as quais, no entanto, não são associadas à resistência a rifampicina. O LPA-plus não identificou resistência à isoniazida em três isolados fenotipicamente resistentes (23/26; 88,5% de sensibilidade) e detectou todos os isolados sensíveis à isoniazida (277/277; especificidade de 100%). Entre os 38 (11%) resultados fenotípicos inválidos, o LPA-plus identificou 31 isolados sensíveis à rifampicina e à isoniazida, um resistente à isoniazida e seis como micobactérias não tuberculosas. Conclusões: O LPA-plus mostrou excelente concordância (≥91%) e acurácia (≥99%). Sua implementação pode acelerar o diagnóstico da tuberculose multirresistente, produzir número significativamente maior de resultados válidos do que o teste fenotípico de suscetibilidade aos antimicrobianos e fornecer informações adicionais sobre o nível de resistência aos fármacos.

Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Phenotype , Rifampin/pharmacology , Time Factors , DNA, Bacterial , Microbial Sensitivity Tests , Prospective Studies , Reproducibility of Results , Sensitivity and Specificity , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Multidrug-Resistant/microbiology , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods , Early Diagnosis , Isoniazid/pharmacology , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/pharmacology
Braz. j. med. biol. res ; 52(3): e8186, 2019. tab, graf
Article in English | LILACS | ID: biblio-989465


Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.

Nucleic Acid Amplification Techniques/methods , Genes, Bacterial , Klebsiella pneumoniae/genetics , Plasmids/isolation & purification , Plasmids/genetics , Temperature , Time Factors , Polymerase Chain Reaction/methods , Reproducibility of Results , Sequence Analysis, DNA , DNA Primers/isolation & purification , DNA Primers/genetics , Limit of Detection , Klebsiella pneumoniae/isolation & purification
An. acad. bras. ciênc ; 90(1): 509-519, Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-886905


ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Gene Expression Regulation, Plant/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Nucleic Acid Amplification Techniques/methods , Saccharum/genetics , Cell Wall/genetics , DNA Primers , Ethanol , Lignin/biosynthesis , Lignin/genetics , Methyltransferases/genetics
Braz. j. microbiol ; 49(1): 128-137, Jan.-Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-889212


ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

Humans , Plague/microbiology , DNA, Bacterial/genetics , Nucleic Acid Amplification Techniques/methods , Magnetics/methods , Yersinia pestis/isolation & purification , Yersinia pestis/classification , Yersinia pestis/genetics , DNA, Bacterial/isolation & purification , DNA, Bacterial/chemistry , Polymerase Chain Reaction , Sensitivity and Specificity , Immunomagnetic Separation , DNA Primers/genetics , Nucleic Acid Amplification Techniques/instrumentation , Magnetics/instrumentation
Clin. biomed. res ; 38(4)2018.
Article in Portuguese | LILACS | ID: biblio-1023783


Introdução: As transfusões sanguíneas começaram a ser realizadas no Brasil no século XX como forma de tratamento terapêutico. Com a descoberta do vírus HIV, a segurança do sangue doado passou a ser prioritária. Assim, candidatos à doação de sangue são submetidos a uma triagem clínica e sorológica, além do teste de ácido nucleico (NAT), obrigatório desde 2014 nos bancos de sangue. Métodos: Estudo retrospectivo através da análise de dados dos doadores de sangue de um Serviço de Hemoterapia em Porto Alegre/RS, nos anos de 2015 a 2017. Avaliando resultados sorológicos e da técnica NAT para HIV. Resultados: Das 28.625 amostras de usuários do serviço de hemoterapia, 41 (0,14%) foram reagentes para o HIV e 21 (0,07%) foram reagente para o teste NAT. Estes dados demonstram uma reatividade duas vezes maior nas amostras de bolsas testadas sorologicamente quando comparadas com a metodologia utilizada no NAT. Conclusão: O avanço científico e tecnológico tem auxiliado no que se refere a redução dos riscos de transmissão de doenças infecto-contagiosa por transfusão sanguínea. O teste NAT teve um acréscimo significativo na pesquisa dos vírus para a segurança na liberação de hemocompoentes. O teste foi introduzido nas rotinas de banco de sangue no intuito de reduzir o período de janela imunológica quando comparado aos testes sorológicos, fato este não observado nos anos de coleta de dados no Serviço de Hemoterapia referido neste estudo. (AU)

Introduction: Blood transfusions began to be performed in Brazil in the twentieth century as a form of therapeutic treatment. With the discovery of the HIV, the safety of donated blood became a priority. Therefore, candidates for blood donation are subjected to clinical and serological screening, in addition to the nucleic acid test (NAT), which has been mandatory since 2014 in blood banks. Methods: We conducted a retrospective study using data from blood donors at one hemotherapy service in Porto Alegre, state of Rio Grande do Sul, from 2015 to 2017. Serological and NAT results for HIV were evaluated. Results: Of the 28,625 samples of users of the hemotherapy service, 41 (0.14%) were HIV reagents and 21 (0.07%) had a reagent result for the NAT test. These data demonstrate a two-fold higher reactivity in the samples of serologically tested units as compared to the methodology used in NAT. Conclusions: Studies with different time periods are needed to further explain this association. The NAT test had a significant increase in the search for viruses and the safety in the release of blood components. The test was introduced in the blood bank routines in order to reduce the window period when compared to serological tests, a fact that was not observed in the years of data collection in the hemotherapy service referred to in this study. (AU)

Humans , Serologic Tests/methods , Acquired Immunodeficiency Syndrome/diagnosis , HIV/isolation & purification , Nucleic Acid Amplification Techniques/methods , Retrospective Studies , Blood Safety/methods
Electron. j. biotechnol ; 28: 113-119, July. 2017. tab, ilus, graf
Article in English | LILACS | ID: biblio-1015986


Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.

Sulfotransferases/metabolism , Pinctada , Nacre/biosynthesis , Chondroitin Sulfate Proteoglycans/biosynthesis , Sulfotransferases/genetics , Nucleic Acid Amplification Techniques/methods , RNA Interference , Real-Time Polymerase Chain Reaction
Biol. Res ; 49: 1-8, 2016. ilus, graf, tab
Article in English | LILACS | ID: biblio-950865


BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.

DNA-Directed RNA Polymerases/genetics , RNA, Viral , Genome, Viral , Sequence Analysis, RNA/methods , Virus Assembly , Nucleic Acid Amplification Techniques/methods , Reference Values , Software , Central African Republic , Reproducibility of Results , Alphavirus/genetics , Mengovirus/genetics , Computational Biology , Contig Mapping
Braz. j. microbiol ; 46(2): 619-626, Apr-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-749730


In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

Animals , Humans , Brucella/isolation & purification , Molecular Diagnostic Techniques/methods , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Nucleic Acid Amplification Techniques/methods , Bacteriological Techniques/methods , Brucella/genetics , Brucellosis/diagnosis , DNA Primers/genetics , Mycobacterium avium subsp. paratuberculosis/genetics , Paratuberculosis/diagnosis , Time Factors
Braz. j. microbiol ; 45(4): 1385-1391, Oct.-Dec. 2014. ilus, tab
Article in English | LILACS | ID: lil-741291


An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

Bacteriological Techniques/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Salmonella typhi/isolation & purification , Typhoid Fever/diagnosis , DNA Primers/genetics , Malaysia , Sensitivity and Specificity , Salmonella typhi/genetics , Time Factors , Typhoid Fever/microbiology
Rev. argent. microbiol ; 46(3): 196-200, oct. 2014.
Article in Spanish | LILACS | ID: biblio-1008778


Las técnicas de amplificación de ácidos nucleicos (NAT) se incorporaron en los bancos de sangre para reducir el riesgo residual de transmisión de infecciones por vía transfusional. La cocirculación de distintas variantes del HIV-1 en Argentina indica la necesidad de evaluar la sensibilidad de los ensayos serológicos y moleculares disponibles para su detección. En este trabajo se evaluó la sensibilidad del equipo COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche), para detectar ARN viral en plasmas de individuos infectados con HIV-1 de Argentina. Los resultados demuestran que esta técnica tiene una alta sensibilidad para detectar ARN de HIV-1 en las condiciones ensayadas: para ensayo de mini-pooles (pooles ≥ 50 copias de ARN/ml), la sensibilidad fue ≥ 92 %, y para procedimiento estándar (plasmas ≥ 207 copias de ARN/ml), la sensibilidad fue 100 %. Además, la técnica COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche), es adecuada para la detección de las variantes de HIV-1 prevalentes

The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreenTM HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreenTM HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants

Humans , HIV-1/isolation & purification , Nucleic Acid Amplification Techniques/methods , HIV Infections/blood
Rev. Soc. Bras. Med. Trop ; 47(4): 418-425, Jul-Aug/2014. tab, graf
Article in English | LILACS | ID: lil-722310


Introduction Previous studies have shown high residual risk of transfusing a blood donation contaminated by human immunodeficiency virus (HIV) or hepatitis C virus (HCV) in Brazil and motivated the development of a Brazilian platform for simultaneous detection of both viruses by nucleic acid amplification test (NAT) denominated HIV/HCV Bio-Manguinhos/Fundação Oswaldo Cruz (FIOCRUZ). The objective of this study was to verify seroprevalence, incidence and residual risk for both viruses before and after the implementation of NAT. Methods Over 700,000 blood samples from all blood banks in the southern Brazilian State of Santa Catarina were analyzed during the period between January 2007 and July 2013. Results Compared with the period preceding the NAT screening, HIV prevalence increased from 1.38 to 1.58 per 1,000 donors, HIV incidence rate increased from 1.22 to 1.35 per 1,000 donor-years, and HIV residual risk dropped almost 2.5 times during the NAT period. For HCV, seroprevalence increased from 1.22 to 1.35 per 1,000 donors, incidence dropped from 0.12 to 0.06 per 1,000 donor-years, and residual risk decreased more than 3 times after the NAT implementation. Conclusions NAT reduced the duration of the immunologic window for HIV and HCV, thus corresponding to approximately 2.5- and 3-fold respective residual risk reductions. .

Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Blood Donors/statistics & numerical data , HIV Infections/epidemiology , Hepatitis C/epidemiology , Nucleic Acid Amplification Techniques/methods , Brazil/epidemiology , HIV Infections/diagnosis , Hepatitis C/diagnosis , Incidence , Mass Screening/methods , Prevalence , Risk Factors
Colomb. med ; 45(2): 61-66, Apr.-June 2014. ilus, tab
Article in English | LILACS | ID: lil-720243


Objective: To compare the diagnostic performance of seven methods to determine Trypanosoma cruzi infection in patients with chronic Chagas disease. Methods: Analytical study, using the case-control design, which included 205 people (patients with Chagasic cardiomyopathy, n= 100; control group, n= 105). Three enzyme linked immunosorbent assays, one indirect hemagglutination assay and one immunochromatographic test were assessed. Additionally, DNA amplification was performed via the PCR method using kinetoplast and nuclear DNA as target sequences. For the comparative analysis of diagnostic tests, the parameters used were sensitivity, specificity, positive and negative predictive values, Receiver Operator Characteristic (ROC), positive and negative likelihood ratio, as well as κ quality analysis. Results: The commercial Bioelisa Chagas test showed the highest sensitivity (98%), specificity (100%), and positive and negative predictive values; additionally it had the highest discriminatory power. Otherwise, the amplification of T. cruzi DNA in blood samples showed low values of sensitivity (kinetoplast DNA= 51%, nuclear DNA= 22%), but high values of specificity (100%), and moderate to low discriminatory ability. Conclusion: The comparative analysis among the different methods suggests that the diagnostic strategy of T. cruzi infection in patients with chronic Chagas disease can be performed using ELISA assays based on recombinant proteins and/or synthetic peptides, which show higher diagnosis performance and can confirm and exclude the diagnosis of T. cruzi infection. The molecular methods show poor performance when used in the diagnosis of patients with chronic Chagas disease.

Objetivo: Comparar la capacidad diagnóstica de siete métodos para determinar infección por Trypanosoma cruzi, en pacientes con enfermedad de Chagas crónica. Métodos: Estudio analítico de casos y controles, que incluyó 205 personas (pacientes con miocardiopatía chagásica, n= 100; grupo control, n= 105). Se evaluaron tres inmunoensayos enzimáticos, una hemaglutinación indirecta y una inmunocromatografia. Adicionalmente, se realizó amplificación de ADN de T. cruzi por reacción en cadena de la polimerasa utilizando como secuencias diana ADN de kinetoplasto y nuclear. Para el análisis comparativo de las pruebas diagnósticas, los parámetros utilizados fueron sensibilidad, especificidad, valores predictivo positivo y negativo, análisis ROC, razón de verosimilitud positiva y negativa, así como análisis de calidad κ. Resultados: La prueba Bioelisa para Chagas mostró la mayor sensibilidad (98%), especificidad (100%) y valores predictivos positivo y negativo; además ésta tuvo el mayor poder discriminatorio. En contraste, los ensayos de amplificación de ADN de T. cruzi mostraron baja sensibilidad (ADN de kinetoplasto= 51%, ADN nuclear= 22%), alta especificidad (100%) y de moderada a baja capacidad discriminatoria. Conclusión: El análisis comparativo entre los métodos sugiere utilizar como estrategia diagnóstica en pacientes crónicos con enfermedad de Chagas, los ensayos de ELISA con proteínas recombinantes y/o péptidos sintéticos por mostrar un rendimiento diagnóstico superior y tener la capacidad de confirmar y descartar el diagnóstico de infección por T. cruzi. Los métodos moleculares muestran pobre rendimiento para ser utilizados en el diagnóstico de pacientes en fase crónica con enfermedad de Chagas.

Adult , Female , Humans , Male , Young Adult , Chagas Cardiomyopathy/diagnosis , Chagas Disease/diagnosis , Trypanosoma cruzi/isolation & purification , Case-Control Studies , Chronic Disease , Enzyme-Linked Immunosorbent Assay/methods , Hemagglutination Tests/methods , Chromatography, Affinity/methods , Likelihood Functions , Nucleic Acid Amplification Techniques/methods , Predictive Value of Tests , ROC Curve , Sensitivity and Specificity
Biol. Res ; 47: 1-10, 2014. ilus, tab
Article in English | LILACS | ID: biblio-950749


BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

DNA, Bacterial/isolation & purification , Nucleic Acid Amplification Techniques/methods , Integrons , Organic Chemicals , Salmonella/genetics , Serratia marcescens/genetics , Staphylococcus/genetics , Vibrio cholerae/genetics , Colony Count, Microbial , Microbial Sensitivity Tests , Polymerase Chain Reaction/methods , Sensitivity and Specificity , DNA, Complementary , DNA Primers , Integrases/genetics , Drug Resistance, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli/genetics , Fluorescent Dyes , Hot Temperature