Multiplex PCR technique
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Multiplex PCR technique
It is a simultaneous method that detects multiple targets in a single reaction. With different reagents for each target. This technique requires two or more tests that can be differentiated from each other and detected simultaneously. The PCR technique is used in life science research, forensic laboratories, and clinical diagnostics. There are two commonly known types of PCR techniques: a quantitative PCR technique. It is a technique in which DNA molecules are tagged using a fluorescent dye, which is used to detect and quantify PCR products in real-time. Reversed transcriptase PCR technique. It is a technique that creates complementary DNA by reversing transcribing RNA to DNA using reverse transcriptase.
Some of the advantages of using multiplex PCR include; it involves few pipetting errors, time-saving, saves on cost due to fewer inputs used, more information with fewer samples, fewer input materials are required, more data from limited starting materials, and improved accuracy from data normalization. However, the PCR technique has some unavoidable demerits, including detecting only a small number of organisms in a sample, contaminations from a repeated number of target sequences leading to contamination of the laboratory environment, and false results that may be caused by a minute amount of carryovers.
Diagnosing of Falciparum Malaria
Falciparum malaria is a bacterial disease that is caused by a parasite called plasmodium falciparum. Plasmodium is a unicellular protozoan parasite that causes malaria in humans.
The PCR technique can be used in detecting the availability of plasmodium in a DNA test. In a way that was pooling the clinical specimens, which could reduce the number of samples needed when screening the disease’s infection. The PCR based samples are to be the most and very important samples and tests to diagnose the infection of malaria disease. We then adopted the pooling system that could now reduce the number of samples that we used.
An example of a test was done to evaluate the tests; two samples of 100 serum are used, with 1% and 5% malaria prevalence. DNA is extracted from the pooled sample amplified by malaria PCR. Additional validation is performed by detecting the level of PCR based on 1:10 and 1:100 solutions. The two tests will detect all malaria samples. Store the serum samples for implications of studying investigating malaria prevalence rates retrospectively. Using the serum and the whole blood specimens are needed to validate these methods’ adaption for clinical utility.
Malaria positive samples are then randomly placed in the two matrices in a blinded fashion. Then rows and columns are made using 100ml serum from each sample. This making it 20 pools of 1ml each. After that, from every pool, DNA is extracted from 400ml of pooled sera using the DNA easy blood and tissue kit according to the manufacturers, blood, and body fluid protocol. The results are as follows, the DNA that was extracted from the 200ml of each undiluted malaria positive serum sample was used as the positive control. Similarly, the DNA from 200ml serum from the case that did not have malaria based on the light microscopy and the PCR technique was then used as the negative control.
In conclusion, the multiplex PCR technique contains both merits and also demerits during its performance; the merits include: the PCR technique contains and gives very broad information with less performed samples, due to use of fewer samples, the PCR technique saves time, the PCR technique also saves on cost as fewer inputs are used, few or no detected pipetting errors as few samples are used, fewer inputs are used during the test thus saving on cost, there is also increased accuracy of data normalization as few samples are used.
However, the multiplex PCR technique contains some unavoidable disadvantages as it is performed. Their demerits include: due to the use of few samples, the PCR technique amplify a small number of genetic materials thus can only detect a few numbers of organisms in a sample as compared to other techniques which use a large number of samples, thus a large number of organisms in a sample, although it has a high specification, this only works under certain conditions, the length of the target DNA sequence is a factor in the level of specification, if the target sequence is too long, then it isn’t easy to ensure that the sequence will occupy in its accuracy.
In conclusion, the multiplex PCR technique is preferred over the other detecting techniques. It is time-saving, minimizes the cost, uses fewer inputs, and is more accurate than other methods.