The overall goal of the qPCR method is to evaluate the viral load of equine herpesvirus-2 in biological samples from horses. This method can help answer key questions in the diagnostic fields of respiratory disease in horses, quantitative data on new interpretative aids for practitioners. The main advantage of this technique is that it is sensitive, specific and a rapid method.
Another advantage is the development according to AFNOR norme NF U47-600, which is the French representative in the international normalization committee. Demonstrating the procedure will be Erika Hue, a young researcher from my unit. To extract nucleic acids from a biological sample of respiratory fluids, under a fume hood begin by adding 140 microliters of biological sample to 560 microliters of lysis solution and incubate at room temperature for 10 minutes.
Add 560 microliters of ethanol to the sample, and apply 630 microliters of the total solution to a silica column and centrifuge. Apply the remaining 630 microliters to the silica column and centrifuge again. After the sample has been applied to the column use 500 microliters each of wash buffers AW1 and AW2 to wash the column two times.
Then to elute the nucleic acids from the column add 50 microliters of room temperature elution or AVE buffer. Close the cap and incubate at room temperature for one minute. Then centrifuge at 6, 000 gs for one minute.
To amplify the DNA, after titrating the primers and probe according to the text protocol, prepare 22.5 microliters of reaction mix for each reaction by adding 12.5 microliters of PCR master mix, 20 micromolar forward and reverse primers, 10 micromolar of the probe, and enough ultra-pure water as required to reach 22.5 microliters. To avoid contamination, always prepare the PCR reaction mix in a specific area. Aliquot 22.5 microliters of the appropriate reaction mix to each reaction well of a 96 well plate.
Include negative controls for extraction and PCR to ensure that none of the reagents are contaminated with unwanted DNA. Next add 2.5 microliters of either the sample, the extraction negative control, the PCR negative control, or the positive control sample to the corresponding reaction wells. Then use an adhesive plate seal to cover the plate.
And centrifuge at 6, 000 gs for 10 seconds. Place the plate in a real-time PCR system. Then select the template for the assay layout and the PCR program settings shown here, and start the run.
After transferring the raw data from the real-time PCR system to a spreadsheet, set the threshold in the amplification plots above the baseline and within the exponential growth region to obtain the threshold cycle for each sample. Plot each point standard set as a standard curve to obtain linearity. Then calculate the copy number for the different samples based on the standard curve.
To determine the limit of detection of the qRT-PCR results, dispense 90 microliters of ultra-pure water into six tubes. To prepare six tenfold serial dilutions of the plasmid to target the abatement zone, begin by transferring 10 microliters from the plasmid working dilution to the tube with 90 microliters of ultra-pure water. Briefly vortex and centrifuge the tube.
Then transfer 10 microliters from the tube to the next tube of water. After vortexing and centrifuging repeat the transfer until all of the tubes of water receive the plasmid. After amplifying the DNA in the six tenfold serial dilutions as described earlier in this video determine the abatement zone, which is the last dilution of plasmid that produces a positive signal and the first dilution without the detection.
To prepare six twofold serial dilutions, dispense 25 microliters of ultra-pure water into six tubes. From the last dilution of plasmid that gave a positive signal from the tenfold dilutions transfer 25 microliters from the tube into the first tube of water. Vortex and centrifuge before preparing the remaining five dilutions as just demonstrated.
Amplify the DNA from the twofold serial dilutions as described earlier in this video. After amplifying the DNA from the three independent trials Calculate the number of positive replicates out of 24 replicates for each level of plasmid concentration. Determine the LOD with a confidence of 95 percent or LOD 95 percent PCR, which is the level that results in the detection of 23 positive replicates out of 24 replicates.
To determine the LOD of the method Prepare five twofold serial dilutions starting with 25 microliters of the plasmid working dilution that is four times more concentrated than the last concentration from a tenfold dilution that gave a positive signal. The plasmid used in the amplification step comes from a plasmid working dilution prepared in a specific area to avoid contamination. It's a critical step.
Transfer 5 microliters from each dilution of plasmid to four tubes with 135 microliters of the negative resource material to obtain four replicates of the five positive standards. Carry out extraction of the four replicates for the five positive standards and perform the amplification procedure as described earlier in this video. Count the number of positive replicates out of eight replicates for each level of plasmid concentration.
Finally, determine the LOD method, which is the last level at which eight replicates out of eight replicates are positive. The quantitative RT-PCR method described in this video detects and quantifies equid herpesvirus-2 in respiratory fluids. In this experiment, tenfold serial dilutions were made to estimate the abatement zone, which lies between dilutions 10 to the negative 9th and 10 to the negative 10th.
The LOD PCR value was determined with a new twofold serial dilution in the abatement zone. To determine the linearity range and LOQ PCR The LOD PCR value was used to start the range of six tenfold serial dilutions between 2.6, the LOD PCR value, and 260, 000 copies per 2.5 microliters of sample. The LOQ PCR is the lowest concentration in the linearity range.
The characterization of the whole method is the validation of all steps necessary to obtain qRT-PCR data from the extraction of DNA from the respiratory sample, to the amplification and quantification of the target. The quantitative performance of the qRT-PCR whole analytical method was evaluated and validated with an accuracy profile represented in this graph. The EHV2 viral genome loads in 172 nasal swab samples from horses with respiratory disorders where quantified as shown here.
The viral genome loads were higher in young horses, with the highest load detected at 1.9 times 10 to the 11th copies per milliliter. Once mastered, this technique can be done in less than two hours per amplification step, and the total of validation steps can be performed in less than four weeks if it is performed properly. While attempting this procedure it's important to remember to avoid risk of contamination particularly when working with a plasmid solution.
Following this procedure, other methods like sequencing can be performed in order to answer additional questions like genome identification. After watching this video you should have a good understanding of how to perform extraction and amplification of DNA and to characterize their PCR reserves. Don't forget that working with biological samples like pathogens can be extremely hazardous.
Some precautions such as working in a hood, and an adapted security level area should always be taken while performing this procedure.
