PCR, as mentioned before, is an acronym for polymerase chain reaction. It is a technique that entails DNA amplification used in detecting the presence of the DNA strands in the infected individuals or organisms.
The requirements for this include three RNA samples which will be used as positive and negative controls. Controls are used to check the accuracy and potency of reagents utilized in the tests. Samples from the suspected chicken will be used for the test. This is a sensitive test that requires accuracy in the measurement of reagents. So, the following reagents are used and are pipetted into the micro-tubes: reverse transcription buffer – 2ul, reverse transcriptase – 1ul, PCR buffer – 5ul, deoxyribonucleotides – 1ul, primer 1 – 1ul, primer 2 – 1ul, sterile water – 37.5ul, and RNA-sample (from chicken) – 1ul.
All these reagents are to be put into the bottom of the PCR micro-tubes before they are put into the thermal cycler. The following conditions are used for a complete PCR: 500C for five minutes, 950C for five minutes, 950C and 550C for thirty seconds each, and 720C for one minute. The long cycle for 10 minutes is to be incubated at 720C. Thus, the whole test will involve three to four hours.
PCR tubes are put into the thermal cycler and are taken out after the reaction is completed. 16ul of the PCR product is taken and put into a new micro-tube, 4ul of 5x gel loading dye into micro-tube, and all 20ul – into the well of agar rose gel.
In order to allow the visualization of the DNA strands on the agar rose gel electrophoresis, DNA fragments should be separated by the use of an electrical current. The gel electrophoresis is done by getting a small amount of prepared agar rose by following manufacturer’s instructions. The gel is then melted in an oven, cooled to a temperature of holding, then gently poured into the gel tray, and allowed to dry before the samples are loaded onto it.
The solidified gel is put into the chamber, and the buffer that is used in electrophoresis is placed to cover the gel assembly; then, the connections are made to run the electrophoresis with the samples loaded onto the gel. This step is important in the visualization of DNA to determine if the PCR process was successful. The DNA strands are stained by the use of the gel loading dye. The dye is closely monitored to allow it flow up three quarters of the gel for approximately 25 minutes. The gels are put into plastic container and methylene blue is poured to stain. Excess dye is rid by submerging the DNA strands on the gel into the tap water. This process is repeated until the banks are visualized, thus it takes around 20 minutes.
PCR products are loaded according to the lines: line 1 – DNA marker, line 2 – positive control, line 3 – negative control, line 4 – suspected chicken 1, line 5 – suspected chicken 2, and line 6 – suspected chicken 3.
Chicken two is infected with avian flu virus. This is because the gene for avian flu virus has been amplified by PCR in the sample obtained from the chicken labeled two.
The lines developed in the test kit are counterchecked to determine corresponds in the appearance, hence diagnosis. The probes appear after staining and can be interpreted as explained in the results.