Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV AP The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice. (C) 2011 Elsevier Ltd. All rights reserved.

Online exercise for the design and simulation of PCR and PCR-RFLP experiments

[EN] Background: Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism of PCR products (PCR-RFLP) are extensively used molecular biology techniques. An exercise for the design and simulation of PCR and PCR-RFLP experiments will be a useful educational tool. Findings: An online PCR and PCR-RFLP exercise has been create that requires users to find the target genes,compare them, design primers, search for restriction endonucleases, and finally to simulate the experiment. Each user of the service is randomly assigned a gene from Escherichia coli; to complete the exercise, users must design an experiment capable of distinguishing among E. coli strains. By applying the experimental procedure to all completely sequenced E. coli, a basic understanding of strain comparison and clustering can also be acquired. Comparison of results obtained in different experiments is also very instructive. Conclusions: The exercise is freely available at http://insilico.ehu.es/edu.

Fish species identification in Canned Tuna by DNA Analysis (PCR-SSCP)

DNA in canned tuna is degraded into short fragments of a rew hundred base pairs. The polymerase chain reaction (PCR) was used to amplify short sequences of mitochondrial DNA, which were denatured and analysed by polyacrylamide gel electrophoresis (native PAGE) for detection of single strand conformation polymorphisms. Species specific patterns of DNA bands were obtained for a number of tuna and bonito species. DE: In Thunfischkonserven liegt die DNA in Form kurzkettiger Fragmente von wenigen Hundert Basenpaaren Länge vor. Mit Hilfe der Polymerase-Kettenreaktion (PCR) wurden kurze Sequenzen der mitochondrialen DNA vervielfältigt. Anschließend wurde die gebildete DNA in Einzelsträngen überführt, die durch eine native Polyacrylamidgel-Elektrophorese (PAGE) aufgetrennt wurde. Für eine Reihe von Thunfischen und Boniten ergaben die Einzelstränge artspezifische Bandenmuster, die auf unterschiedliche Konformationen der DNA-Stränge der einzelnen Fischarten zurückzuführen sind.

Production of positive controls for calcivirus-specific PCR using recombinant baculiovirus technology

Recent advances in our knowledge of the genetic structure of human caliciviruses (HuCVs) and small round-structured viruses (SRSVs) have led to the development of polymerase chain reaction (PCR)-based molecular tests specific for these viruses. These methods have been developed to detect a number of human pathogenic viruses in environmental samples including water, sewage and shellfish. HuCVs and SRSVs are not culturable, and no animal model is currently available. Therefore there is no convenient method of preparing viruses for study or for reagent production. One problem facing those attempting to use PCR-based methods for the detection of HuCVs and SRSVs is the lack of a suitable positive control substrate. This is particularly important when screening complex samples in which the levels of inhibitors present may significantly interfere with amplificiation. Regions within the RNA polymerase regions of two genetically distinct human caliciviruses have been amplified and used to produce recombinant baculoviruses which express RNA corresponding to the calicivirus polymerase. This RNA is being investigated as a positive control substrate for PCR testing, using current diagnostic primer sets. Recombinant baculovirus technology will enable efficient and cost-effective production of large quantities of positive control RNA with a specific known genotype. We consider the development of these systems as essential for successful screening and monitoring applications.

White spot syndrome virus (WSSV) transmission risk through infected cooked shrimp products assessed by polymerase chain reaction (PCR) and bio-inoculation studies

The aim of the study was to evaluate the resistance of white spot syndrome virus (WSSV) in shrimps (Penaeus monodon) to the process of cooking. The cooking was carried out at 1000C six different durations 5, 10, 15, 20, 25 and 30 min. The presence of WSSV was tested by single step and nested polymerase chain reaction (PCR). In the single step PCR, the primers 1s5 & 1a16 and IK1 & IK2 were used. While in the nested PCR, primers IK1 &IK2 – IK3 & IK4 were used for the detection of WSSV. WSSV was detected in the single step PCR with the primers 1s5 and 1a16 and the nested PCR with the primers IK1 and IK2 – IK3 & IK4 from the cooked shrimp samples. The cooked shrimps, which gave positive results for WSSV by PCR, were further confirmed for the viability of WSSV by conducting the bio-inoculation studies. Mortality (100%) was observed within 123 h of intra-muscular post injection (P.I) into the live healthy WSSV-free shrimps (P. monodon). These results show that the WSSV survive the cooking process and even infected cooked shrimp products may pose a transmission risk for WSSV to the native shrimp farming systems.

Molecular probes and polymerase chain reaction (PCR) -based kits for diagnosis of shrimp diseases

Technology for effective and fast diagnosis of animal diseases is essential for developing aquaculture management strategies. This paper reviews the conventional techniques for shrimp disease diagnosis and discusses the emergence of nuclei acid probes and polymerase chain reaction (PCR)-based kits as powerful tools for rapid and accurate detection of shrimp diseases.