HELINI White spot Syndrome virus [WSSV] Real-time PCR Kit
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1 HELINI White spot Syndrome virus [WSSV] Real-time PCR Kit Instruction manual Cat. No: /50/100 tests Compatible with: Agilent, Bio-Rad, Roche, Applied Bio systems [ABI], Rotor-gene, Cepheid, Bioer, and Spartan Real time PCR machine. V version Page 1 V version Page 2
2 Contents Kit components 5 Storage 5 Introduction 7 Pathogen information 7 Principle 8 Material required 11 Protocol Detection mix 13 Amplification Protocol 15 Interpretation of results 16 Limitations 18 V version Page 3 V version Page 4
3 Kit components No. of reactions Probe PCR Master Mix 200µl 2 x 200µl Taq Enzyme Mix 50µl 2 x 50ul WSSV Primer Probe Mix 65µl 2 x 65µl Internal control Primer Probe Mix 65µl 2 x 65µl Internal control template [IC template] 125µl 2 x 125µl Water, PCR grade 4ml 4ml WSSV QS1 [Positive control Standard] 125µl 125µl Instruction manual 1 1 Storage The content of the kit should be stored at 20 C and are stable until the expiration date stated on the label. Repeated thawing and freezing (>2 x) should be avoided, as this may reduce assay sensitivity. If the reagents are to be used only intermittently, they should be frozen in aliquots. Storage at 2 8 C should not exceed a period of 5 hours. Intended Use The is an in vitro nucleic acid amplification kit for the detection of WSSV virus in Shrimp biological samples. This kit is compatible with all Real time PCR machines. [ABI, Agilent, Roche, Rotor gene, Bio-Rad, Cepheid, Bioer and Spartan] Product Use Limitations All reagents may exclusively be used in Molecular Diagnosis. The product is to be used by personnel specially instructed and trained in Molecular diagnosis. Strict compliance with the user manual is required for optimal PCR results. Attention should be paid to expiration dates printed on the box and labels of all components. Do not use expired components. Technical Assistance For technical assistance and more information, please contact; helinibiomolecules@gmail.com Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. Discard sample and assay waste according to your local safety regulations. V version Page 5 V version Page 6
4 Introduction HELINI White sport syndrome virus [WSSV] Real-time PCR Kit constitutes a ready-to-use system for the detection of WSSV virus using polymerase chain reaction (PCR). It contains reagents and enzymes for the specific amplification of 106bp region of the WSSV genome, and for the direct detection of the specific amplicon in fluorescence channels Cycling Green. In addition, it contains an internal control amplification system to identify possible PCR inhibition. External positive controls (WSSV Positive Template) are supplied, which can be used as both qualitative and quantitative to determination the amount of viral load. Specificity WSSV primer and Probe have been designed for the specific and exclusive in vitro quantification of WSSV virus. The target sequence (Ribonucleotide reductase large unit) is highly conserved and has previously been shown to be a good genetic marker for WSSV. The primers and probe sequences in this kit have 100% homology with a broad range of relevant reference sequences based on a comprehensive bioinformatics analysis. Dynamic range of test Under optimal PCR conditions, the kit have very high priming efficiencies of >95% and can detect less than 10 copies of target template. Positive control For copy number determination and as a positive control for the PCR set up, the kit contains a positive control template. This can be used to generate a standard curve of copy number / CT value. Alternatively the positive control can be used at a single dilution where full quantitative analysis of the samples is not required. Each time the kit is used, at least one positive control reaction must be included in the run. A positive result indicates that the primers and probes for detecting the target pathogen gene worked properly in that particular experimental scenario. If a negative result is obtained the test results are invalid and must be repeated. Care should be taken to ensure that the positive control does not contaminate any other kit component which would lead to false-positive results. This can be achieved by handling this component in a Post PCR environment. Care should also be taken to avoid cross-contamination of other samples when adding the positive control to the run. This can be avoided by sealing all other samples and negative controls before pipetting the positive control into the positive control well. The quantitation standards are defined as IU/μl. The following equation has to be applied to convert the values determined using the standard curve into copies/ml of sample material: Result (Copies/μl) x Elution Volume (μl) Result (Copies/ml) = Sample Volume (ml) As a matter of principle, the initial sample volume should be entered in the equation above. V version Page 7 V version Page 8
5 Negative control To confirm the absence of contamination, a negative control reaction should be included every time the kit is used. For this reaction, the RNAse/DNAse free water should be used instead of template. A negative result indicates that the reagents have not become contaminated while setting up the run. If a positive result is obtained the results should be ignored and the test samples repeated. Possible sources of contamination should first be explored and removed. Internal Control template When performing DNA/RNA extraction, it is often advantageous to have an exogenous source of nucleic acid template that is spiked into the lysis buffer. This internal control nucleic acid template is then co-purified with the sample DNA/RNA and can be detected as a positive control for the extraction process. Successful copurification and real-time PCR for the control template also indicates that PCR inhibitors are not present at a high concentration. The primer and probe present at PCR limiting concentrations which allows multiplexing with the target sequence primers. Amplification of the Internal control template does not interfere with detection of the pathogen target gene even when present at low copy number. The Internal control is detected through the HEX channel and gives a CT value of 26 +/-3. Material Required 0.2 ml PCR tubes / 8 well strips / 96 well plate according to real time PCR machine & Model. Micro Pipettes Variable Volume μl, μl, and μl Sterile pipette tips with aerosol barrier 2-20μl, μl, and μl Disposable powder-free gloves Centrifuge with 1,5ml tubes rotor 1.5ml/2ml centrifuge tubes Precautions for PCR Store positive material (Specimens, Standards or amplicon) separately from all other reagents and add it to the reaction mix in a separate facility. All the positive standards & specimens should be mixed & dispensed in extraction area. All the reagents including the NTC (except for standards & specimens) should be mixed & dispensed in pre-mix area. Use pipette tips with filters only. Always use disposable powder-free gloves Add 5μl of the internal control template to each sample. Do not add directly to sample. Add to sample/lysis buffer complex. Complete purification according to the manufacturer s protocols. V version Page 9 V version Page 10
6 Preparation of standard curve dilution series: 1. Pipette 450µl of Nuclease free water into three 1.5ml micro centrifuge tubes and label QS2 to QS4. 2. Pipette 50µl of WSSV QS1 Positive Template into tube QS2. 3. Vortex thoroughly and spin down briefly. 4. Change pipette tip and pipette 50µl from tube QS2 into tube QS3. 5. Vortex thoroughly and spin down briefly. 6. Repeat steps 4 and 5 to complete the dilution series. 7. Use 10µl per reaction. Copy Number Standard curve copies per µl WSSV QS WSSV QS WSSV QS WSSV QS Detection Protocol Things to do before starting Before each use, all reagents need to be thawed completely, mixed by gently inverting and centrifuged briefly. Make sure that Positive and Negative control is included in every PCR run. Make sure that internal control template is added during DNA purification. Detection Mix Components Volume Probe PCR Master Mix 8µl Taq Enzyme mix 2µl WSSV PP Mix IC PP Mix Purified DNA sample* 2.5µl 2.5µl 2 to10µl Total reaction volume 25µl Note*: DNA purified from Gills use 10µl of elute. Other samples use 2 to 5µl of elute & make up the final reaction volume to 25µl with sterile water. Centrifuge PCR vials briefly before placing into thermal cycler. V version Page 11 V version Page 12
7 Negative Control setup [NTC] Add 10µl of nuclease free water. Qualitative Positive Control setup Add 10µl of any one of the Positive controls [From QS1 to QS4] Quantitative Positive controls setup Include all Positive controls prepared from QS1 to QS4. Amplification Protocol Step Time Temp Reading the graph: Step-1 Select only test sample wells, select HEX/VIC/JOE/Cy3 channel and view the graph of internal control amplification. A successful amplification Ct value must be less than Ct 31. [Range 16 to 31]. This range indicates that test sample is purified well and there is NO PCR inhibition in the reaction. Any sample value goes beyond Ct value 31 indicates that either sample has some issues in the purification or inhibiting PCR reaction. 45 cycles Taq enzyme activation 15min 95ºC Denaturation 20sec 95ºC Annealing/Data collection* 20sec 56ºC Extension 20sec 72ºC Only one exceptional case that Ct value beyond 31 can be accepted that the test sample is high positive for the WSSV. Any test sample Ct value is beyond 31 must be re-purified and re-run the assay. Step-2 Select the NTC and Positive control [Qualitative] or Standards wells [Quantitative], select FAM channel, view the graph of amplification. WSSV = FAM channel Internal Control = HEX Channel The NTC must be flat with no Ct value. If required adjust the threshold value just above the NTC. The PC or Standards must be amplified as per their copy numbers. NTC justifies NO contamination in the reagent as well as fine pipetting and its environment. V version Page 13 V version Page 14
8 PC justifies the reagents storage conditions and reaction parameters are as prescribed. Step-3 In FAM channel, Select the NTC well and test sample well one by one, analyze the graph/amplification. Every time, select the NTC well and the test sample well, one by one analyze. Interpretation of Results Negative control The Negative control reactions should not exhibit fluorescence growth curves that cross the threshold line. If a false positive occurs with one or more of the NTC reactions, sample contamination may have occurred. Invalidate the run and repeat the assay with stricter adherence to the procedure guidelines. Positive Control Positive control reactions should produce positive results before 36 cycles. If expected positive reactivity is not achieved, invalidate the run and repeat the assay with stricter adherence to the procedure guidelines. Test Sample/Specimen - Positive When all controls meet stated requirements, a test sample/specimen is considered presumptive positive. Test Sample Negative control Positive control Internal Control Interpretation Positive Negative Positive Positive Detected Negative Negative Positive Positive Not-Detected Negative Negative Negative Negative Positive Positive Positive Positive Internal control - Interpretation Experiment fail Experiment fail When used according to the above protocols, (assuming 100% extraction efficiency) CT value is expected within 21 to 31. However, this can vary significantly depending on the extraction efficiency, the quantity of elute added to the PCR reaction and the individual machine settings. CT values of 26±5 are within the normal range. When amplifying sample with a high genome copy number, the internal control may not produce an amplification plot. This does not invalidate the test and should be interpreted as a positive experimental result. Test sample/specimen - Negative When all controls meet the stated requirements, sample is considered negative. V version Page 15 V version Page 16
9 Limitations A false negative result may occur if inadequate numbers of organisms are present in the sample due to improper collection, transport or handling. A false negative result may occur if an excess of DNA template is present in the reaction. If late CT or inhibition of the Internal control is noted for a particular sample, purified DNA can be tested at 2 or more dilutions [e.g., 1:5 and 1:10) to verify the results. In association with G Gautham Pulavar Molecular Research Manufactured and Marketed by HELINI Biomolecules, Ohmlina, 26, 2 nd Avenue, Khuthubi Complex, Vettuvankeni, Chennai , Tamilnadu, INDIA helinibiomolecules@gmail.com Analysts should be trained and familiar with testing procedures and interpretation of results prior to performing the assay V version Page 17 V version Page 18
10 V version Page 19 V version Page 20
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