This practical activity was carried out to establish an unknown species of malaria using the molecular method and clinical specimens. The choice for the molecular method to determine this particular malarial species is because the traditional approaches are less precise and efficient in identifying parasitemia in low concentration or when there is a co-occurrence of another infection. Besides, traditional approaches require skilled professionals to determine the species correctly. The study aimed at specific loci of 18s rRNA gene for amplification. A fresh blood sample used in the test was collected from a patient in EDTA tube for Malarial Nested PCR. The study used species-specific Oligonucleotide primers, a universal reverse primer and forward primer for the subunit ribosomal RNA to form a 783-821 bp product. After this step, the final PCR products are run on an agarose gel through electrophoresis after which positive controls are used to contrast the band lengths which helps identify the malarial species in the patient blood.
To avoid contamination, sterilization measures are undertaken all through the process as well as the use of evidence-based control measures. The practical activity used tow control and the negative control which ensured that there is enough sterilization in the whole process. The human control primers are used in the experiment to assess the presence of any inhibitors which can deter human PCR amplicons from amplifying which could hinder them from being visible in the agarose gel. The equipment used in the experiment were sterilized well before their use.
Approach and materials used
Positive control preparation
The practical analysis used blood that had been collected in clinical cases. The EDTA blood collected was diagnosed with malaria through microscopy test previously and stored at -20 degrees Celsius. The blood used in the experiment was pooled from different patients with the aim of pooling different species of malaria in the pool of blood used to carry out the analysis and with a good concentration of organisms, i.e., 100 organisms per cubic of measurement. The positive control is treated with other test specimens after which DNA is extracted in agreement with the specimen processing manual. A series of dilution is carried on the samples with the aim of monitoring inhibition.
First-round malaria PCR amplification master mix
The application of master mix in the samples helps eliminate any possible pipetting recurrence and abatements. This activity reduces the number of defrost cycles of the used reagents which reduces the time taken to carry out the procedure and further reduces any the risk of contamination of the experiment solution.
First-round PCR amplification mix
- 0.2ml PCR tubes are used to process the samples and dispense the reactions.
- The first round master mix should be prepared in Ice in the following sequence;
i. AmpliTaq Gold DNA polymerase 0.50 l/tube
ii. Round 1 malaria MM 44.50 l/tube
- Pipette 45ul of the master mix to every tube, vortex, and centrifuge the mix before transferring it to the ice tray to add the sample.
- dispense 5ul of the sample to increase its volume to 50ul in the following order;
i. Undiluted sample
ii. Carry out a No DNA control to assess the presence of any possible contamination of the PCR sample.
- This step is followed by a vortex and centrifuge of all the samples followed by removing them from the cabinet. The positive control should be taken out from the freezer.
- The tubes with samples then should be pipetted using 5ul of positive control. The tubes should be centrifuged at 14000rpm for ten seconds and then transferred to the thermal cycler for the initial amplification.
The First-round of thermal amplification profile
This cycle is necessary for the experiment because it enables the PCR to extend and lengthen after which it is cooled by the thermal cycler to 250C and stored in a refrigerator until it is ready to be analyzed using agarose gel electrophoresis.
The Second-round Malaria PCR amplification master mix
This step uses 2ul from the first round malaria PCR amplification which is diluted using grade water at the ratio of 1:100. The resulting component is used in the second amplification cycle. However, master mixes are used that contain reverse primers, and they are specific to every malarial species.
Second-round amplification thermal profile
Steps to be followed:
- Establishment of 9 racks of 0.2 ml PCR reaction tubes which should be labeled.
- Every malarial species is assigned to three 0.2 ml tubes. That is, three tubes of 0.2 ml PCR for P. falciparum, P. Vivax, and P. ovale respectively. The master mixes are labeled PF MM, PV MM, and PO MM respectively.
- From each species master mixes samples of testing are chosen and assigned to test; 1 unknown, 1 negative and 1 positive control.
- 0.8ul is added to FAL
- 91.2ul of the master mix is added to FAL
- 0.8ul taq is added to VIV containing test tube
- 91.2ul of Master Mix is added to VIV test tube
- 0.8 ul taq is added to OVA test tube
- 91.2 of Master Mix is added to OVA test tube
- 23 ul of FAL should be added to 0.2 ml test tubes labeled 1, 2 and 3 and the lid should be closed tightly.
- 23 ul of VIV should be added to 0.2 ml tubes 4, 5 and 6 and also closed tightly.
- 23 ul of OVA should be added to 0.2 ml tubes 7, 8 and 9
- 2 ul of UNK sample should be added to tubes 1, 4 and 7
- 2 ul of NEG sample to be added to tubes 2, 5 and 8
- 2 ul of POS sample should be added to tubes 3, 6 and 9
- All test tubes should be sealed tightly after which Pulsed centrifuge of the tubes should be done.
- After the centrifuge, the samples are put in a thermo cycler for the second round of amplification stage.
- After the thermal cycler stage is completed the tubes should be removed to run them on the agarose gel electrophoresis.
Agarose Gel Electrophoresis (AGE)
At this stage, 2.0 percent agarose gel is required and put in 0.25 tubes. 2.0ul containing GLB III which is a loading buffer is mixed with 10ul of sample. After this activity, 8ul of the molecular standard should be dispensed in the mix and placed in 100 base pair ladder for reference. The gel should be run in a 200 vortex for 50 minutes, and ethidium bromide added to stain the DNA on the agarose gel. After finishing these activities, a UV light photo is taken, and the bands analyzed.
Results and discussions
After carrying the experiment, it was concluded that a positive sample for malaria should have a band which is almost the same size as the positive control. The samples from the test that did not have a band similar to the positive control were perceived to have tested negative to malaria. The PCR amplicons presence in NDC indicated that there was contamination during the PCR creation steps. From the observation of the gel, the positive control worked, and the band is of the expected size. Also, the negative control did not have any band which evidenced that the negative control was good for the study. The first tube had a weak band which was concluded as a result of faulty loading of the sample, but it did not match the band size of P. falciparum which is a negative result. The fourth well has a clear band that has a perfect match with the positive control of P. vivax, and the seventh well had a weak band that does not match with the band size of P. ovale which excluded the ovale malarial species.
It can be concluded that the results were accurate and all the controls worked well as it was anticipated in the theoretical framework. The results found out that the well 4 tube band matched perfectly with the positive control for P. vivax which concludes that the unknown malarial species in the sample is P. vivax.
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