, Diagnosis an-&& of Shrimp Diseases - Central Institute of ...

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A typical amplification reaction includesI. The sample of the target DNA11. Two oligonucleotide primers111. Four Deoxynucleotide triphosphates (dNTPs)IV. Reaction bufferV. Magnesium and optional additivesVI. Taq-DNA polymeraseVII. dd Hz0The components of the reaction are mixed and placed in an automated instrumentcalled thermocycler that takes the reaction to a series of different temperatures for varyingamounts of time. This series of temperature and time is referred to as one cycle ofamplification. In each cycle of amplification the quantity of target DNA doubles, and as fewas 20 cycles would generate approximately a million times the amount of target DNA whichwas present initially.The first step of PCR involves thermal denaturation of the double-stranded targetDNA molecules. The next step is the annealing of oligonucleotide primers to thecolnplementary target sequences by temperature reduction. Th,~reafter, prilner directed DNAsynthesis reaction will follow with the help of thermostable DNA taq polymerase, resulting inthe doubling of the amount of target sequence in the sample. By repeating the cycle ofdenaturation, primer annealing and DNA synthesis (primer extension), the copy number ofthe target DNA is increased exponentially.Once a product is obtained, it can be analyzed in a number of ways like agarose gelelectrophoresis. The products will be readily visible by UV transillumination of an ethidiumbromide stained gel.Nested PCRNested PCR or two-step PCR is useful in reducing or eliminating unwanted productssilnulta~leously increasing the sensitivity significantly. An aliquot of the first PCR product isthen subjected to an additional round of amplification using primers complementary to thesequences internal to the first set of primers. Only the legitimate product is amplified in thesecond round. This approach of two-step amplification is often successful even if the.designed product is initially below the level of detection by ethidium bromide staining.Optimization of PCRDesigning of an ideal primer pair, optimisation of the concentration of Mg, primer andtemplate DNA, buffer pH and cycling conditions are important for the success of a PCR.Ideally PCR primers should have 40 - 60 % G+C content, which generally range in length
from 15-30 bases. Negative controls are mandatory in each PCR run to rule out any false-positive results caused by contamination. However, every step should be taken to avoid thepossible contamination during the setting up of a PCR.Nucleic Acid ProbesNucleic acid probes are segments of DNA or RNA that have been labelled withenzymes, antigenic substances, chemiluminescent substances or radioisotopes. Probes can bedirected to either DNA or RNA targets. Probes can bind with complimentary sequences ofpathogenic DNA during the detection process providing a signal (like colour change) that canbe identified or measured. Nowadays, non-radioactive probes (eg: digoxigenin (DIG) labelledprobes) are gaining importance due to their high level of sensitivity and safety as compared tothe radioactive probes. The in-situ hybridization and dot blot hybridization are examples ofgene probes, which are finding its use in aquatic disease diagnostics. However, PCR hasadvantages over the gene probes in its sensitivity to be used for direct detection in clinicalspecimens.PCR - based diagnosis of Indian white spot virusIn the aquaculture of penaeid shrimps, White Spot Disease (WSD) caused by WhiteSpot Virus (WSV) is the major cause of morbidity and mortality today in Asia, resulting inhuge economic losses for shrimp farmers. Among all the recorded viral diseases in culturedpenaeids, WSD is the most widespread. The rapid onset and lethality of WSV has put Asianshrimp farming as a wliole at the breaking point. The WSV was first reported in 1992 fromTaiwan, subsequently from most shrimp farming countries in Asia and recently from thewestern hemisphere.White spot disease has diminished the prospects of shrimp farming in India. This isthe most virulent virus ltnown to affect cultured shrimps. Till date, no treatment is known tocontrol the White Spot Disease. Hence, early diagnosis followed by suitable managementpractices is the only alternative in tackling this disease. Diagnosis of WSD can be done bymethods such as histopathological techniques. A presumptive diagnosis can also be done byobserving clinical symptoms such as the presence of white spots. These methods can detectthe WSD only in the late stage of infection. The PCR is a powerful and sensitive diagnostictools for identification of viral pathogens even at a very early stage (asymptomatic / carrierstage) of infection.
Page 5 and 6:
OVERVIEW OF STRATEGIES FOR PREVENTI
Page 7 and 8:
in harmonizing aquatic animal healt
Page 9 and 10:
SI-IRXMP DISEASES: GENEIL4L ASPECTS
Page 11 and 12: quantitative level of pathogen is i
Page 13 and 14: presence of prominent eosinophiiic
Page 15 and 16: Disease affected the shrimps of all
Page 19 and 20: BACTERIAL AND FUNGAL DISEASES OF SH
Page 21 and 22: Cause: The epibiotic bacteria stich
Page 23 and 24: PARASiTIC AND NON-INFECTIOUS IIISEA
Page 25 and 26: XOS-INFECTIOUS DISEASESNon-infectio
Page 27 and 28: INVESTIGATING SHRIMP DISEASES: OBSE
Page 29 and 30: lnay be a predisposing factor for b
Page 31 and 32: BACTERIOLOGICAL METHODSS.V. Alavand
Page 34 and 35: In place of ZMA, nutrient agar prep
Page 36 and 37: Interpretation: Positive reaction i
Page 38 and 39: Incubate at 37°C for 1-2 h.Examine
Page 40 and 41: Alkaligens: etc. are resistant. The
Page 42 and 43: Culture Media, Reagents and StainsC
Page 45 and 46: Differential characteristics of Vib
Page 47 and 48: Davidson's Alcohol Formatin Acetic
Page 49 and 50: Paraffin cold impregnationXylene an
Page 51 and 52: ProcedureDe-paraffinise the slid'c
Page 53 and 54: Biochemistry of Nucleic Acids with
Page 55 and 56: DNA e.rl1i6it.v base equivuleizcc!E
Page 57 and 58: hydrolyzed by dilute alkali. The su
Page 59 and 60: Polymerase Chain Reaction (PCR) is
Page 61: a number of viral, bacterial and pa
Page 65 and 66: PCR Primers for Indian WSVTwo sets
Page 67 and 68: ANTIOXIDANTS IN MANAGEMENT OF OXIDA
Page 69 and 70: Many potent chemical carcinogens ar
Page 71 and 72: cells. It has been shown that possi
Page 73 and 74: AFB, induced ~nutagenesis in Salmon
Page 75 and 76: FIGURE 2. An overview of oxygen rad
Page 77 and 78: were responsible for production los
Page 79 and 80: for Asiatic region. The participati
Page 81 and 82: charge of the DNA fragment. The neg
Page 83 and 84: European countries have in place th
Page 85: irth to a new concept to treatment
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