ABSTRACT
La papa (Solanum tuberosum) Diacol Capiro es uno de los cultivares con mayor producción y consumo interno en Colombia, siendo los departamentos de Cundinamarca y Boyacá, los principales productores. Este cultivo, se ve afectado por un complejo de virus, que disminuye la calidad de los tubérculos y los rendimientos. En este trabajo, se evaluó la prevalencia de los virus de ARN: PLRV, PVY, PVX, PVS, PVV, PYVV, PMTV y PVB, en brotes de tubérculos-semilla certificados, provenientes de la sabana Cundiboyacense, mediante RT-qPCR. Los resultados revelan la ocurrencia de siete de los ochos virus en las muestras, con niveles de infección de 100 % (PVS, PVX y PYVV), 75 % (PLRV), 50 % (PVY), 37,5 % (PMTV) y 12,5 % (PVB). Adicionalmente, con el fin de obtener información de los genomas de los virus detectados, se utilizó secuenciación de alto rendimiento (HTS), de una muestra compuesta (bulk) de brotes, siendo posible obtener el genoma completo del PLRV y el genoma parcial del PVY. Los análisis filogenéticos realizados con dichas secuencias ubicaron a los virus PLRV y PVY en clados, conformados por aislamientos colombianos, con niveles de identidad superiores al 97 %. Estos hallazgos evidencian la necesidad de fortalecer los programas de certificación de tubérculos-semilla de papa en el país, mediante la utilización de pruebas moleculares de detección viral.
Diacol-Capiro is one of the most important potato (Solanum tuberosum) cultivars in Colombia with most production concentrated in the provinces of Cundinamarca and Boyacá. Unfortunately, this crop is seriously affected by several viruses that compromise the quality of tubers and yields. In this work, it was evaluated the prevalence of the RNA viruses: PLRV, PVY, PVX, PVS, PVV, PYVV, PMTV, and PVB in certified tuber-seed sprouts produced in the highlands of Cundinamarca and Boyacá by RT-qPCR. Results revealed a prevalence of 100 % for PVS, PVX, and PYVV; 75 % for PLRV, 50 % for PVY, 37.5 % for PMTV, and 12.5 % for PVB. Additionally, high-throughput sequencing from a sprout´s bulk sample was used to gather genomic information of infecting viruses, which resulted in a partial PVY sequence, and a complete PLRV genome. Phylogenetic analysis revealed that both assemblies cluster within clades comprising other Colombian isolates with more than 97 % nucleotide sequence identity. These findings highlight the need to update potato seed-tuber certification programs in Colombia with the implementation of more sensitive molecular tests.
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Objective To develop a real-time RT-PCR assay ( rRT-PCR) for efficient detection of duck hepatitis virus type 1 ( DHV-I) .Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay ( rRT-PCR) .Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates.The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China.Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.
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Objective To investigate the infection situation of HIV in patents in hospital ,and to provide basis for the prevention of AIDS in the hospital .Methods Three kinds of enzyme‐linked immunosorbent assay(ELISA)kits(Beijing wantai ,Beijing kewei , French BIO‐RAD)were used for screening HIV antibody and antigen in 240 781 samples from Jan .2011 to Dec .2013 .All HIV re‐peated positive screening samples were confirmed by western blotting in Chongqing shapingba Center for Disease Control and Pre‐vention .Results Among 240 781 samples from 2011 to 2013 ,593 samples(0 .246% ) were HIV positive at the first screening ,558 samples(0 .231% ) were HIV positive in at the repeated screening ,29 samples(0 .012% ) were HIV indeterminate ,6 samples(0 . 002% ) were HIV negative.Male and female ratio was 3 .39:1 .Conclusion Screening in hospital patients could be an important way to discover cases with HIV infection .It is nesscessary to strengthen the promotion and propaganda of HIV detection ,and new technology of HIV detection could be used to strengthen the inspection of HIV .Moreover ,the consciousness of self protection should be promoted in the treatment of HIV patients .
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A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV)strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.
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In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.
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BACKGROUND: Respiratory viral infections can become epidemic due to high contagiosity. Since there was no rapid diagnostic method for complete diagnosis in the past, diagnosis was solely made on the basis of clinical symptoms or the time of infection. With recent developments in rapid diagnostic methods like multiplex reverse transcriptase (RT)-PCR, R-mix virus culture, etc., early detection and effective treatment of respiratory viral infections is possible. Herein, we compared the efficiency of multiplex RT-PCR and the R-mix virus culture for the rapid detection of respiratory viruses. METHODS: We used 96 nasopharyngeal swab specimens for culturing respiratory viruses using R-mix (Diagnostics Hybrids Inc., USA). Afterwards, multiplex RT-PCR was performed using specimens stored at -70degrees C. RESULTS: R-mix virus culture yielded positive results in 34 cases (35.4%) and multiplex RT-PCR in 73 cases (76.0%). Both methods yielded identical results in 51 cases (29 positive cases and 22 negative cases). Among 45 cases that showed different results, 40 showed negative results in R-mix virus culture and positive results in multiplex RT-PCR, and 1 showed positive result in R-mix virus culture and negative result in multiplex RT-PCR. Different viruses were detected in the remaining 4 cases by both the methods. CONCLUSIONS: Multiplex RT-PCR provided faster results and had higher detection rates than R-mix virus culture. Further, unlike R-mix virus culture, multiplex RT-PCR can be used to identify new respiratory viruses. Therefore, multiplex RT-PCR is more useful than R-mix virus culture in the diagnosis of respiratory virus infection.
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , RNA, Viral/analysis , Reagent Kits, Diagnostic , Respiratory Tract Infections/virology , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , Virus Cultivation , Virus Diseases/diagnosis , Viruses/geneticsABSTRACT
Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.
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The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20 percent of the AdV, 90 percent of the RV and 100 percent of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8 percent of the AdV and 90 percent of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.
Subject(s)
Adenoviruses, Human/isolation & purification , RNA Viruses/isolation & purification , Sewage/virology , Water Microbiology , DNA, Viral/isolation & purification , Hepatitis A virus/isolation & purification , Poliovirus/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , RNA, Viral/isolation & purification , Rotavirus/isolation & purificationABSTRACT
PURPOSE: Although the nasopharyngeal aspirate (NPA) is more commonly used because of relatively higher accuracy, the nasal swab (NS) is a less painful and easier method than NPA. A few recent reports have shown that NS is more effective than NPA for the detection of respiratory virus using immunofluorescence or viral culture. The objective of the present study was to compare the results of NPA and NS sampling specimens in children for respiratory viruses detection using multiplex RT-PCR. METHODS: From December 2008 to June 2009 Paired NPA and NS specimens were collected from 250 children admitted with symptoms of acute respiratory infections at the Department of Pediatrics, Soonchunhyang University Cheonan Hospital, Cheonan, Korea. The sensitivity and agreement of virus detection between NPA and NS using multiplex RT-PCR were compared and analyzed. RESULTS: The median age of the subjects was 1.3 years (range, 20 days to 16.5 years), and 228 patients (91.2%) were under the age of 5 years. The agreement of virus detection between NPA and NS was excellent (Cohen's kappa >0.8) for parainfluenza virus type 3 or substantial (0.6 to 0.8) for rhinovirus A, RSV A and RSV B, moderate (0.4 to 0.6) for adenovirus and metapneumovirus and poor (<0.4) for influenza A. The overall sensitivity of detection of respiratory viruses was significantly higher in NPA (0.96) than in NS (0.59, P<0.05). CONCLUSION: We recommend NPA may be more accurate specimen for detection of respiratory viruses in hospitalized children. NS might be used in limited cases at a office setting or for larger epidemiological studies. However, results obtained from NS for influenza virus type A, metapneumovirus and adenovirus, should be interpreted carefully.
Subject(s)
Child , Humans , Adenoviridae , Child, Hospitalized , Epidemiologic Studies , Fluorescent Antibody Technique , Influenza, Human , Korea , Metapneumovirus , Orthomyxoviridae , Parainfluenza Virus 3, Human , Pediatrics , Respiratory Tract Infections , Rhinovirus , VirusesABSTRACT
PURPOSE: Although the nasopharyngeal aspirate (NPA) is more commonly used because of relatively higher accuracy, the nasal swab (NS) is a less painful and easier method than NPA. A few recent reports have shown that NS is more effective than NPA for the detection of respiratory virus using immunofluorescence or viral culture. The objective of the present study was to compare the results of NPA and NS sampling specimens in children for respiratory viruses detection using multiplex RT-PCR. METHODS: From December 2008 to June 2009 Paired NPA and NS specimens were collected from 250 children admitted with symptoms of acute respiratory infections at the Department of Pediatrics, Soonchunhyang University Cheonan Hospital, Cheonan, Korea. The sensitivity and agreement of virus detection between NPA and NS using multiplex RT-PCR were compared and analyzed. RESULTS: The median age of the subjects was 1.3 years (range, 20 days to 16.5 years), and 228 patients (91.2%) were under the age of 5 years. The agreement of virus detection between NPA and NS was excellent (Cohen's kappa >0.8) for parainfluenza virus type 3 or substantial (0.6 to 0.8) for rhinovirus A, RSV A and RSV B, moderate (0.4 to 0.6) for adenovirus and metapneumovirus and poor (<0.4) for influenza A. The overall sensitivity of detection of respiratory viruses was significantly higher in NPA (0.96) than in NS (0.59, P<0.05). CONCLUSION: We recommend NPA may be more accurate specimen for detection of respiratory viruses in hospitalized children. NS might be used in limited cases at a office setting or for larger epidemiological studies. However, results obtained from NS for influenza virus type A, metapneumovirus and adenovirus, should be interpreted carefully.
Subject(s)
Child , Humans , Adenoviridae , Child, Hospitalized , Epidemiologic Studies , Fluorescent Antibody Technique , Influenza, Human , Korea , Metapneumovirus , Orthomyxoviridae , Parainfluenza Virus 3, Human , Pediatrics , Respiratory Tract Infections , Rhinovirus , VirusesABSTRACT
Objective Effects of different factors on shoot tip culture in vitro were studied by using Dioscorea bulbifera as test material to find the media suitable to the shoot tip differentiation of D.bulbifera and the method of virus delection.Methods Plant tissue culture method was used in shoot tip culture and the symptom observation method,instruction plant method,and RT-PCR method were used in virus detection of virus-free plantlets.Results The best disinfection method for D.bulbifera shoot tips was firstly disinfected for 30 s with 70% alcohol and then disinfected for 12 min with 0.1% HgCl2;It is better for D.bulbifera to cut shoot tips in 0.5 mm length after 37 ℃ heat treatment for 7 d;The best proliferation medium of D.bulbifera shoot tips was MS+KT 2 mg/L+NAA 0.5 mg/L;The best rooting medium of regeneration buds from D.bulbifera shoot tips was 1/2 MS+NAA 0.5 mg/L;The best matrix of regeneration plantlets from D.bulbifera shoot tips was perlite-vermiculite (2∶1);RT-PCR Method was primary mean to detect potato virus Y (PVY) of regeneration plantlets from D.bulbifera shoot tips,and average rate of PVY-free was 86.5% by RT-PCR.Conclusion The virus-free culture system of D.bulbifera shoot tips is established for the first time,providing a technological basis for the rapid propagation and factory production of D.bulbifera virus-free plantlets.