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.

Study of white spot disease in four native species in Persian Gulf by histopathology and PCR methods

After serious disease outbreak, caused by new virus (WSV), has been occurring among cultured penaeid shrimps in Asian countries like China since 1993 and then in Latin American countries, during June till July 2002 a rapid and high mortality in cultured Penaeus indicus in Abadan region located in south of Iran with typical signs and symptoms of White Spot Syndrome Virus was confirmed by different studies of Histopathology, PCR, TEM, Virology. This study was conducted for the purpose of determination of prevalence(rate of infection)/ROI and grading severity (SOI) of WSD to five species: 150 samples of captured shrimps and 90 samples of cultured ones; Penaeus indicus, P. semisulcatus, P. merguiensis, Parapenaopsis styliferus, and Metapenaeus affinis in 2005. 136 of 240 samples have shown clinical and macroscopical signs & symptoms including; white spots on carapase (0.5-2 mm), easily removing of cuticule, fragility of hepatopancreas and red color of motility limbs. Histopathological changes like specific intranuclear inclusion bodies (cowdry-type A) were observed in all target tissues (gill, epidermis, haemolymph and midgut) but not in hepatopancreas, among shrimps collected from various farms in the south and captured ones from Persian Gulf, even ones without clinical signs. ROI among species estimated, using the NATIVIDAD & LIGHTNER formula(1992b) and SOI were graded, using a generalized scheme for assigning a numerical qualitative value to severity grade of infection which was provided by LIGHTNER(1996), in consideration to histopathology and counting specific inclusion bodies in different stages(were modified by B. Gholamhoseini). Samples with clinical signs, showed grades more than 2. Most of the P. semisulcatus and M. affinis samples showed grade of 3, in the other hand in most of P. styliferus samples grade of 4 were observed, which can suggest different sensitivity of different species. All samples were tested by Nested PCR method with IQTm 2000 WSSV kit and 183 of 240 samples were positive and 3 1evel of infection which was shown in this PCR confirmed our SOI grades, but they were more specified.

Rapid detection of white spot syndrome virus (WSSV) of Penaeus monodon by latex agglutination test using monoclonal antibodies

Latex beads were sensitized with monoclonal antibodies (MAb) rose against VP28 of WSSV. The optimum concentration of MAb required to sensitize the latex beads was 125 µg/ml. The sensitized latex beads were used to detect WSSV from PCR-positive stomach tissue homogenates obtained from infected shrimp. Stomach tissue homogenates from WSSV-infected shrimp agglutinated the sensitized latex beads within 10 minutes, while uninfected samples did not produce any agglutination, although non-specific agglutinations were observed in some samples. The analytical sensitivity, analytical specificity, diagnostic sensitivity and diagnostic specificity of the (LAT) agglutination test were assessed. The analytical sensitivity of the test was 40 ng of purified WSSV (2 µg/ml). The sensitized latex beads did not agglutinate with normal shrimp tissue or MBV-infected tissue homogenate. The test has a diagnostic sensitivity of 70 and 45%, respectively, compared to single-step and nested PCR. The diagnostic specificity of the test was 82%. This test is a simple and rapid on-farm test which can be used to corroborate clinical signs for the detection of WSSV in grow-out ponds.

Survey of Amphiprion clarki & Amphiprion sebae populations in Persian Gulf by molecular genetics

Anemone fishes are a group of 28 species of coral reef fishes belonging to the family Pomacentridae, subfamily Amphiprioninae and all have an obligate symbiotic relationship with sea anemones. Two species of these small ornamental fishes have been identified in the Persian Gulf including Amphiprion clarkii and A. sebae. The phylogenetic relationship between Amphiprion species of the Persian Gulf was studied by collecting 15 samples from three Iranian islands, Larak, Farur and Kish. DNA was extracted from each sample and a part of mtDNA was amplified. Two pairs of primers were designed to amplify a final target of 400 by nested-PCR. Each amplicon was sequenced, aligned and genetic diversity among samples was investigated by phylogenetic analysis. Results show that there is no significant genetic variation among A. clarkii individuals; however, A. sebae individuals from Larak were different from other fishes of the same species. Most probably this is due to the ability of A. clarkii to be symbiotant with all 10 species of host sea anemones which enables it to spread its own population in the 3 islands. However, A. sebae is observed to be symbiotant only with one host in the sea, therefore, has one option that reduces its distribution.

Development and evaluation of the polymerase chain reaction (PCR) method for diagnosis of spring viremia of carp virus (SVCV)

Aquaculture has been expanded rapidly to become a major commercial and food-producing sector worldwide in recent decade. In parallel, viral diseases rapidly spread among farms causing enormous economic losses. The accurate detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Spring Viraemia of Carp Virus (SVCV) is a very severe pathogen of carp fishes in different parts of the world and is categorized as a reportable listed disease in the annual published list of World Organization for animal Health (OIE). The objective of this study was to develop and evaluate RT- PCR test for detecting SVC virus and also the sensitivity and specificity of this test. A semi nested RT- PCR was designed using combination of three primers: two external (SVCF , SVCR) and one internal (SVCS) primers which based on conserved region of G gen. The specificity of designed primers (only external ones) by examination on Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) was confirmed. For optimizing of the PCR test, primer concentration, primer annealing temperature, cycle number and Mgcl2 concentration were surveyed. Also for validity test, prevention of false negative and Assurance of its accuracy, a competitive internal control (mimic) designed and its suitable concentration was defined. Evaluation of the sensitivity of designed test were conducted first by comparing the different commercially available RNA isolation guidelines, two guidelines: isotiocyanate phenol–chloroform based protocols (RNX–Plus Iran, Iq2000 kit Taiwan ) and two column based protocols (Cinna pure RNA Iran , high pure viral RNA kit, Roche Germany ). The results indicated that the column based protocols (Roche method and Cinna pure), yield 36.77 ng/μl and 16/47 ng/μl RNA concentration respectively, which were significantly higher than other protocols(P<0.05). Then for evaluation of extracted RNA sensitivity, Serial dilution of SVCV strain 56.70 grown in EPC (1.9×105 TCID50/ml) was examined To compare sensitivity. Extracted RNA from serial dilution with stone's primers and commercial IQ-2000 kit were examined simultaneously. The result indicated that designed semi- nested RT- PCR was able to recognize SVC virus to 10-4 dilution and stone's primer recognize to 10-3 dilution whereas Iq-2000 commercial kit did not recognized in any dilution. In high virus titer in designed test two DNA band (462 bp and 266 bp) produced, and by decreasing virus titer 462 bp was omitted. In low virus titer or lack of virus, just DNA band (mimic) 729 bp can propagate. After designing and optimizing PCR test, a total of 400 suspected cultured Cyprinus carpio with high mortality from 4 aquaculture zone of Khuzestan province were collected and tested for SVCV during 2012- 2013 using developed PCR method and IQ- 2000. The results indicated that SVC virus was not observed in samples using both methods.

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.

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.

PCR-SSCP and Isoelectric Focusing as Screening Methods for Identification of (Sub-) Tropical Fish Species

To ensure the authentication of fishery products lacking biological characters, rapid species identification methods are required. Two DNA- and protein-based methods, PCR-SSCP (polymerase chain reaction - single strand conformation polymorphism) of a 464 bp segment of the cytochrome b – gene and isoelectric focusing (IEF) of water-soluble proteins from fish fillets, were applied to identify fillets of (sub-) tropical fish species available on the European market. Among the samples analysed were two taxonomically identified species from the family Sciaenidae and one from Sphyraenidae. By comparison of DNA- and protein patterns of different samples, information about intra-species variability of patterns, and homogeneity of batches (e.g. fillet blocks or bags) can be obtained. PCR-SSCP and IEF may be useful for pre-checking of a large number of samples by food control laboratories. Zusammenfassung Zur Sicherstellung der Authentizität von Fischerei-Erzeugnissen ohne biologische Merkmale sind schnelle Verfahren zur Speziesidentifizierung hilfreich. Zwei Methoden der DNA- bzw. Protein-Analyse wurden eingesetzt, um Filets (sub-) tropischer Fischarten, die auf dem europäischen Markt angeboten werden, zu identifizieren. Bei diesen Methoden handelt es sich um die PCR-SSCP (Polymerase-Kettenreaktion – Einzelstrang-Konformationspolymorphismus) – Analyse der PCR-Produkte und die IEF (isoelektrische Fokussierung) der wasserlöslichen Fischmuskelproteine. Unter den untersuchten Proben waren zwei taxonomisch bestimmte Arten aus der Familie Sciaenidae und eine Spezies aus der Familie Sphyraenidae. Durch Vergleich der DNA- bzw. Proteinmuster lassen sich Informationen über die intra-spezifische Variabilität solcher Muster und die Einheitlichkeit von Partien (beispielsweise Filetblöcke oder Filetbeutel) gewinnen. PCR-SSCP und IEF können in Laboratorien der Lebensmittelüberwachung als Vortest gerade bei hohen Probenzahlen sinnvoll eingesetzt werden.

PCR-based assay for detection of four coral pathogens

Several microorganisms have been identified as pathogenic agents responsible for various outbreaks of coral disease. Little has been learned about the exclusivity of a pathogen to given disease signs. Most pathogens have only been implicated within a subset of corals, leaving gaps in our knowledge of the host range and geographic extent of a given pathogen. PCR-based assays provide a rapid and inexpensive route for detection of pathogens. Pathogen-specific 16S rDNA primer sets were designed to target four identified coral pathogens: Aurantimonas coralicida, Serratia marcescens, Vibrio shilonii, and Vibrio coralliilyticus. Assays detected the presence of targets at concentrations of less than one cell per microliter. The assay was applied to 142 coral samples from the Florida Keys, Puerto Rico, and U.S. Virgin Islands as an in situ specificity test. Assays displayed a high-level of specificity, seemingly limited only by the resolution of the 16S rDNA.

Investigation of polymorphism by PCR-RFLP method in Cobia fish Rachycentron canadum in the Persian Gulf and Oman Sea

Cobia is a native fish species in Iranian waters in the Persian Gulf and Sea of Oman and has a good internal and foreign market. This fish is a fast growing species and for this reason Iranian Fisheries is considering to go for it culture practices. To go for any utilization such as fishing from wild stocks or culture activities, needs a better understanding of its peculiarities and genetic characteristics of its natural resources. Therefore, this project was discribed and conducted. In this investigation, cuts 2 or 3 cm of fin tissue of  specimen of Cobia obtained from Sistan and Bluchestan, Hormozgan, Bushehr and Khuzestan water provinces, were collected. DNA was extracted by Phenol-chlorophorm method and produced PCR product in length of 1060 and 1450 base pair of two mitochondrial genes COI and NADH2. Using 13 cutting enzymes (4 enzymes were subscriber for both of genes), 205 base pair (from 2510 base pair, equal with %3.8 from gene regains) were directly investigated. But binding patterns of enzymatic digestion of PCR products of both COI and ND genes from electrophoresis were monomorph in all samples and no polymorphism was observed. This may be attributed to the unsuitable choice of COI and ND2 genes for showing of intra specific divergence. But in general non-existence of genetic diversity or noticeable decrease of that among individuals has been reported in regions were fish migration exist and they can freely move between two regions. Therefore, non-observation of polymorphism in the study area might be the case and indicates represents the area. On the other hand, some scientists believe that the distributions of populations in different regions are greatly affected by environmental and physical and ecological factors. Althoug Cobia is a migratory fish, but with regard to the fact that the environmental conditions are different (specially temperature and salinity) between east and west of Persian Gulf and Oman sea, there is a possibility that different genetic groups of this species exist in the regions. Of course It is clear that using more samples and enzymes from other genetically regions could produce better results. Since none of the two investigated genes didn’t show genetic divergence or polymorphism amongst the individuals of one region or between different regions, therefore, statistic analysis for estimating of haplotype diversity or nucleotide diversity and drawing of relationship tree among individuals using available softwares was not possible.

Genetic variation survey of intra-specific (Sepia pharaonis) in the Persian Gulf and the Oman Sea using PCR-RFLP

This research was conducted to identify Cuttlefish population (Sepia pharaonis) in The Persian Gulf and the Oman Sea using PCR-RFLP. Specimens were collected from )0 different stations. Bottom trawling method was used for sampling from different zones of the Persian Gulf and the Oman Sea, and finally specimens from S. Pharaonis were collected at each station . DNA was extracted by phenol—Coloroform method. One pair primer was designed based on 1As rRNA gene nucleotide sequences. The results obtained from 1 As rRNA gene RFLP, which was reproduced by PCR technique, were analyzed and utilized for study of diversity of the Cuttlefish population. PCR product with o pair base in length achieved for all specimens, which was subjected to enzymatic digestion by A restriction action enzymes: Alu I-Taq I-Mnl I-Rsa I-Hind III-Dra I-vu II and Hae II DNA bands patterns in all specimens digested by those enzymen showed similarity with no any polymorphism. From this result, it can be concluded that there is not any possibility to isolate different populations in the studied Cuttlefish species under exploitation of rRNA gene.