An experimental report on real-time polymerase chain reaction (PCR) detection of HIV viral load
Experimental rationale
It is important to note that HIV as become a very big menace to society that is why this experiment was quite significant in the sense that, its aim was to generate a real time detection of HIV viral load, a move that is vital for the correct diagnosis and monitoring of HIV in patients.
The aim of the experiment was achieved with the detection of the HIV viral odd using the PCR at 5ml and 4ml from 20ml of cDNA from PCR reaction.
Results
Standard curve
Figure 1. (Standard curve for the student raw data 1)
Table 1. (Patients information table 1)
| WellCт logCopy number/4µl Copy number/mlA114.15717316633977.215583397721.558 B114.56657791634959.786993495978.699 C118.56108475537122.169493712216.949 D119.02514648538050.292973805029.297 E123.32660294437322.56473732256.47 F123.62878418437806.054693780605.469 G127.43050385332916.604613291660.461 H126.75717545332108.610533210861.053 A233.80178452227041.427612704142.761 B234.15726471227325.811772732581.177 C238.34202576115336.81031533681.03 D236.68024826114672.09931467209.93 E2Undetermined0#VALUE!#VALUE! F2Undetermined0#VALUE!#VALUE! G2Undetermined#VALUE!#VALUE! H2Undetermined#VALUE!#VALUE! |
Figure 2. (Standard curve for the student raw data 2)
Table 2. (Patients information table 2)
| Sample Name | log | Cт | Copy number/5µl | Copy number/ml | |
| Sample 1A | 6 | 20.14251137 | 60427.5341 | 6042753.41 | |
| Sample 1A | 6 | 19.8387146 | 59516.1438 | 5951614.38 | |
| Sample 1B | 5 | 25.01685524 | 62542.1381 | 6254213.81 | |
| Sample 1B | 5 | 24.98981857 | 62474.54643 | 6247454.643 | |
| Sample 2A | 4 | 15.68968868 | 31379.37737 | 3137937.737 | |
| Sample 2A | 4 | 15.70296764 | 31405.93529 | 3140593.529 | |
| Sample 2B | 3 | Undetermined | #VALUE! | #VALUE! | |
| Sample 2B | 3 | Undetermined | #VALUE! | #VALUE! | |
Real-time PCR data charts
Figure 3
Amplification plot
Figure 4
Melt curve
Experimental limitations
As an investigative tool, real- time polymerase chain reaction is vital in clinical molecular diagnostics because of its ability to generate real-time, accurate measurements of viral and in most instances, bacterial loads. In this case, the main goal of real-time polymerase chain reaction (PCR) was to accurately differentiate and measure specific HIV viral load sequences in the given samples even if there was a very tiny quantity of HIV viral loads in the samples (Busby and Garson, 2017).
The advantage
The application of real- time PCR in the detection of the HIV viral load came with several advantages but the most distinct advantage was in its ability to quantify the volumes of HIV viral loads over a large dynamic range. The other advantage is that real-time polymerase reaction was performed in a closed reaction medium that required no post- PCR handlings there reducing the chance for cross- contamination of the samples in the lab
The limitation
Even with these advantages of real- time PCR methodology it is important to note that some limitations were encountered chief among them being that, inhibitors were found in the body fluids such as haemoglobin (Moustafa and Cross 2018).
Concluding sentence
The application of real-time PCR in the detection of HIV viral load is a clear testimony to the ability and influence of real-time PCR in expounding molecular events that underlie human physiology.
Optional extensions
Brief background information on HIV/AIDS
Infections involving the Human immunodeficiency virus (HIV) are the sources of the acquired immunodeficiency syndrome which in most cases, is referred to as AIDS. The HIV virus attacks body cells which are responsible for bolstering the body’s immune system and subsequently, impairs the immune system’s ability to defend the body against other disease attacks. In most cases, the virus is transmitted through direct contact with an infected body fluid for example, blood and semen or in some cases through contact with an infected mucosal membrane. After the completion of an initial asymptomatic phase, the first symptoms of HIV/AIDS begin to manifest in form of skin problems, extreme loss of weight and upper respiratory tract infections among others (Hubert and Sandfort, 2019).
Consideration of other techniques used for disease diagnosis or monitoring
The universal surveillance of diseases has led to the innovation of a wide variety of techniques used for diagnosing and monitoring diseases and they include:
Branched DNA (bDNA) test.
Nucleic acid sequence-based amplification (NASBA) test.
List of reference
Busby, E. & Garson, J. A. (2017). Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR. Scientific reports, 7(1), 1209.
Hubert, M., & Sandfort, T. (2019). Sexual behaviour and HIV/AIDS in Europe: Comparisons of national surveys. Routledge.
Moustafa, K., & Cross, J. M. (2018). Genetic approaches to study plant responses to environmental stresses.
