Development of a novel quantitative PCR analysis method for HIV-1

Abstract

Biological pharmaceuticals are the answer to many severe diseases of today’s society. Some of these are protein-based drugs and can be produced from human blood plasma. In order to guarantee the safety of patients, absence of any pathogens needs to be ensured. Such a pathogen is HIV-1, which holds high mutation rate and a large variation in its genome.
The aim of this project was to develop novel primers and probes for detection of a wide range of subtypes of HIV-1, using the TaqMan qPCR detection system. Three targets within conserved parts of the genome were selected in the regions of LTR5´, Pol and LTR3´. The primers and probes were optimized regarding concentrations, salt content and compatibility.
The sensitivity of detection showed promising results with a value as low as 8.18 IU. The selectivity did not result as preferred, with the best combination of primers and probe possible to detect 7 out of 8 subtypes of the most common genotype, M. If combining the primers and probes of all targets suggested here, detection of all tested subtypes was possible.
Summarizing the results, the primers and probe targeting the Pol region shows promising data. Optimization regarding the sequences of the Pol primers and probe and additional evaluation of compatibility between all targets need to be studied, in order to see, if the method can meet the standards and further be implemented into the experimental routine for HIV-1 detection.

Popular Abstract

Popular science summary – The potential of better detection of HIV
Viruses have become a global health issue and people are constantly dying from their effects, both in rich and poor countries. This has awakened a new dimension in the urgency and interest of finding new methods of identifying infected people in order to help, treat and stop the spread of infection in the society. In order to make the world a better place for all with a better life quality.
More than 700 000 people died last year of the effects of HIV, and millions are living with the disease causing a poor life quality, worldwide. In order to be able to stop the spread of infection more accurate methods of detecting the virus are necessary. Containing the spread of the illness is paramount, and by making the contagious individuals aware of their infection, the spread will be easier to control as well as the infected individual can get treatment faster. However, many viruses of this kind cannot be cured, proper inhibitor medicines are available. Many of these treatments today have shown promising results with best effect upon usage in early stages of infection. Viruses mutate frequently, in order to avoid the immune system. This makes them a hard target, since they are changing their genetic code constantly. The numbers of infected cells can differ between individuals and in the stage of infection, which puts high standards on detection methods. Two aspects of virus detection are especially important; the possibility of detecting the virus with high specificity, able to distinguish the HIV infected cells from others, and with high sensitivity, capable of finding every infected cell even in low numbers is of great importance.
The work done during the project has shown interesting results of HIV detection. A method using genetic nucleotide sequences has been developed. The developed genetic sequences verifying the starting point of replication in DNA, called primers. Additionally, with a complementary nucleotide sequence to the DNA template, dyed with signal giving elements attached, called probes. The results suggest a detection method with high sensitivity, able to find viruses in very low concentrations within blood. In addition to, good ability of specificity, with as many as 7 out of 8 types of the most common kind of HIV, with the use of 4 replicates. In total the most promising method developed managed to find HIV in 19 out of 24 samples evaluated with prominent signals. These results indicate a highly promising method. At this stage it needs further development in order to meet the standards of European Medical Agency and Food and Drug Administration, as well as being even more precise in specificity.
Substantial theoretical knowledge of HIV and insights in possibilities of detection of the virus has been the foundation to success in the project. Different parameters such as suitable components, time, temperature, target sequences in the genetical code of the virus has been evaluated and optimized in order to develop a most positive method. Both with high specificity and low sensitivity. The aim of using the method in routine analysis is not completely reached, but the method is on the right track to fulfil the demands of it.
The research and development of new detection methods for viruses such as HIV is of great importance to the society. Infected individuals need to know about their disease and being able to get proper treatment as well as not pass the virus further on to their fellow humans. The methods can also be used to certify that donated blood is absent from pathogens. If so, the blood can be donated to those in need or used to produce biological and protein-based pharmaceuticals for severe diseases.

• author: Hallberg, Anna-Malin
• supervisor: Lei Ye; Rasmus Gustafsson
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20202
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences; Technology and Engineering; Chemistry
• keywords: HIV, PCR, primers, probes, virus, detection, blood plasma, pathogen, biological pharmaceutical, protein-based pharmaceutical, genotype, subtype, RT qPCR, extraction, TaqMa, RNA virus, cross-dimer, applied biochemistry, tillämpad biokemi
• language: English
• id: 9037098
• date added to LUP: 2021-01-22 11:26:08
• date last changed: 2021-01-22 11:26:08