Biomedical Science Lab Report

Biomedical Science Lab Report

Student’s Name

Institution

Viruses are microscopic living parasitic organisms that are a bit smaller than bacteria. They are unable to live on their own. Therefore, they attach to another living organism, especially like a human, to reproduce and thrive. Predominantly, viruses are known to cause serious medical complications in human life. The black side of the virus is that they easily lead to death. For instance, the outbreak of Ebola in 2014 from West Africa and swine flu of 2009 that widespread throughout the globe. Recently, the outbreak of Corona Virus (Covid19 Virus) in 2019 that have made the whole globe to remain at a standstill is one of the representations of how dangerous virus is in peoples live. The worst part of it, diseases caused by the virus are difficult to treat, and even getting the vaccine for the same is really hectic.

As viruses host themselves in human beings, they teeter themselves in regions regarded as life. On the one hand, the virus occupies the nucleic acids (DNA and RNA) that makeup living organisms. Ideally, the virus will attach to RNA or DNA (Svintradze, 2017). However, the virus has no ideal capacity to read the codes independently in RNA and DNA. Secondly, the virus is very hard to be noticed due to its microscopic nature. However, their impact on people’s life can be seen easily. When it is attacked by a virus, it is very hard to tell the virus unless quality testing and experiment is done (Isel et al., 2016). For biomedics, this is one of the areas they can specialize in the testing of viruses. The procedure includes taking the samples in the laboratory and conducting the necessary experiments.

The aim of this experiment was to identify which viruses were available in the samples. The importance of detecting which viruses are in a sample or in an individual is to know the medication procedures to be initiated. The potential viruses in this sample are Influenza A and Influenza B, Influenza B, and RSV or Influenza A and Influenza B. However, this will not be substantiated without clear experimentation to determine which among these viruses are in the samples.

Methodology and Results

Extraction of PCR Products

In this session, we started by extracting sample gels that will be used in the experiment to investigate the availability of the potential Viruses. Agarose gel containing 2% off w/v to create bands. The bands were allowed to run for 30 minutes. Fourteen wells were provided to each to help in loading PCR products and provided with equal lanes of the ladder as well. Also, we were provided with 25 microliters of PCR to load to each gel. From these, five microliters were required to be added in each sample and a microliter to be used in loading each dye. (0.25% (w/v)).

The procedure of the extraction of PCR Products

50 microliters of 1Xtbe were added in the gel. After loading, the mixture was run through the electrolysis chamber for approximately 30 minutes, with the electricity current maintained at 100 volts. To ensure the results are correct, the marker was maintained a stable point to prevent running to the end of the gel. The process was monitored closely to see the movement of the dye through the gel. When loading, we ensured that every individual group member was set to prevent his gel. Caution was taken to prevent the staring of the gel without the samples of all other group members. After loading the sample gel, we added five microliters of the DNA ladder to almost the middle of the gel.

Extraction of RNA and RT-PCR

The procedure involves equipment and protocols necessary in the purification of Viral RNA. The initial volume used was 50 microliters. We followed all the required steps necessary to generate cDNA from the RNA template. We used 5 micrograms of purified RNA together with random hexamines to help in priming cDNA synthesis.

The procedure was as follows

The solution to be used was thoroughly vortex and briefly centrifuged before using it. We then prepared the priming prefix by referencing the units of measurements presented to us in table 1. To obtain clear results, the priming premix had to be prepared using the ice in the RNase free reaction tube. The reaction was then mixed gently by the use of a pipette. Pipette ensured that the solution thoroughly mixed but without interfering with solution temperature in case there was extreme mixing. After mixing, the solution was kept in incubation at 25 degrees for exactly 10 minutes. Afterward, the solution was put in the same incubation at 45 degrees for 30 minutes. To finish the reaction, the solution was incubated for 85 degrees for 5 minutes then hold at 4 degrees. After this, we stored the solution for future use in the experiment at -20 degrees. This was to ensure that no bacteria or new virus can enter at low temperatures as bacteria survive at normal body temperature.

To complete this experiment, the following primer designs were used.

After obtaining the primers, we conducted a laboratory test to determine whether the named viruses were available in the solution. From the word go, we identified Influenza A virus to be available in the samples. Therefore, the whole experiment was to determine if Influenza A virus was available.

We pipetted 15 microliters of master mix from the previously prepared PCR placed in an MM tube. After producing multiplex PCR, we merged all primers into a single testing tube. We later on added 8 microliters to al tubes containing PCR prime mergers, and we marked them. After completing this procedure, all our tubes had the needed primers dNTPs, Taq, Water, and buffer all equal to 23 microliters. With our cDNA, we had prepared earlier and stored in incubation at -20 degrees we added it to each PCR tube of 23microliters master mix. Plus, the 2 microliters of cDNA, the total volume was 25 microliters.

Sequencing

We used the Hartwell Center of Biotechnology to determine the sequencing of the DNA by the use of oligonucleotides cycles together with AmpliTaq DNA polymerase.

Results and Observation

Genes Targeted in the PCR and the respective primers

Most of Influenza viruses comprises eight vRNA segments with a polarity of negative. The virus has a genome of A/PR/8/34 (H1N1) that is 13,588 nucleotides long. The genome is divided into several sections. Polymerase genome, both PB1, and PB2 are made up of 2341 nucleotides each. A nucleotide has 1565, Matrix gene 1027, nonstructural 890, and HA has 1778 nucleotides. From the primers obtained, the region of passive coding is made of 5 termini with vRNA made of 20-58 long nucleotides (Nadia & Sadok, 2017). The length of 3 lies between the 19^th and 45^th nucleotides. As seen from the sequencing, the difference is existing within these segments, which are classified into two parts. The first classification is made of 13 stored nucleotides located at each 5’end and the other conserved at 12 nucleotides at 3’-end. All these nucleotides are stored in each vRNA to all influenza viruses.

Substitution is a necessary analysis that is done to any primers found in the 14 or 15, which is found in the 3 ends similar to that found in the 21 (5′-end) terminal nucleotide. The PCR temperatures remained to anneal. To control this, we added the 5^th sequence primer of the noninfluenza virus. This is because this design allows primer amplification for different segments as they differ by as little as 2 or three nucleotides at the 3′-end. We, later on, performed the RT-PCR two-step analysis. The first analysis involved a 12 Uni primer, which is a 12 complimentary of the stored nucleotides at the vRNA conserved at the 3-end. The final step was that of PCR, which included a reverse-transcription of products that were amplified using the primers from segment-specific like Protein nucleotides.

Internal gene analysis of RT-PCR

In this analysis, we tried to determine whether the difference between primers could allow amplification of a specific full-length of the corresponding gene. We, therefore, performed RT-PCR, which had specific primers located at each segment (Doris et al., 2017). The results are shown below.

The diagram represents the preserved terminals in the regions of 3 and 5 across all eight segmentations of Influenza A vRNA together with all universal primers. All the segmentations differ in length but have similar characteristics of segmentation. As observed earlier, all segmentation in 13’ vRNA conserved nucleotides has 5 termini to ever influenza virus. For 13 segmentation is made of 5′ terminus. The segmentation has the Oligo-V sequence that is necessary for mRNAs for polyadenylation. The dash region shows regions of non-conformity.

Results for gel Electrophoresis

The full-length amplification if

The figure shows the photo taken from the electron microscope as a representation of the electrolyzed gel. The full-length amplification included the total tubes filled with the gel containing the virus. The electrolyzed PCR was isolated to help and reverse transaction processes performed to collect Uni12 primer. Subsequently, the electrophoresis process was done to all unknown samples, DNA ladder, and even to the positive controls. Almost 10% of the PCR produced was subjected to direct electrophoresis that contained 1% of agarose gel. To make results more clear, we added ethidium bromide.

Discussion and Conclusion

Choosing the right primer sequences is an essential variable to determine the specificity of viruses in the given samples based on the RT-PCR methods. For this report, we tried d to show how segmentation of all samples containing the virus can be amplified using the RT-PCR method by using several primers (Universal). First, all three lines indicated the presence of universal primers at that is seen at the 3′ end. Available viruses could, therefore, be isolated in one of the samples. Secondly, the database indicated a series of segmentation in different terminus that are found in lane 3 and 5. Our primers included NP, PA, PB2, and PB1. All these had G-residue containing M, HA, and NS primers. Third, we conducted mutational analyses. From this analysis, we found that vRNA was being promoted to secondary structure with a confirmation of a double formation to each specific position like in the region 12 and 13.

The complementary of 12 and 13 mer oligonucleotides is well described by Adeyefa and coauthors by the use of the multiplex RT-PCR method. This resulted in the amplification of the eight regions of RNA (Deubel & T, 2017). However, their primers differ from the primers we used in our experiment. Our primers allowed amplification of the segments fully to ensure the potential virus is clearly seen under an electronic microscope. We based our RT-PCR on the 12^th and 13^th conserved nucleotides. However, the cloning of these segments could be impossible to give us the real virus available in the samples. To overcome this drawback, we used primers we developed through electrophoresis and added them to each specific segment, which helped in the amplification of the intended segment. Gel electrophoresis was used to separate the subtypes of PCR like HA and NA. Zou tried to use different sizes of primers in his experiment. However, our experiment was strictly for 3′ and 5′ length of each specific region that contained the sequences of non-influenza virus (Deubel & T, 2017). Therefore, the presence of sequence analysis worked well in our experiment to help identify the right virus in the experiment.

The virus described above has the following characteristics. The virus has eight segmentation with nucleotides ranging from 12 and 13. The length of the strands ranges from 3’ and 5’ in length. Also, we found the available virus to contain nucleoprotein, nonstructural proteins. The primers of the available virus did not work well under cloning. All these characteristics reflect only one class of viruses; the Influenza A virus.

References

Deubel, V., & T, F. (2017). Virus Identification – an overview | ScienceDirect Topics. Www.Sciencedirect.Com. https://www.sciencedirect.com/topics/medicine-and-dentistry/virus-identification

‌ Doris, P. A., Hayward-Lester, A., & Hays, J. K. (2017). Q-RT-PCR: data analysis software for measurement of gene expression by competitive RT-PCR. Bioinformatics, 13(6), 587–591. https://doi.org/10.1093/bioinformatics/13.6.587

‌ Isel, C., Munier, S., & Naffakh, N. (2016). Experimental Approaches to Study Genome Packaging of Influenza A Viruses. Viruses, 8(8), 218. https://doi.org/10.3390/v8080218

‌ Nadia, B., & Sadok, S. (2017). Comparison of DNA-extraction Methods Suitable for PCR-based Applications to Identify Shrimp Species in Commercial Products. Journal of FisheriesSciences.Com, 11(4). https://doi.org/10.21767/1307-234x.1000138

‌ Svintradze, D. V. (2017). Geometric Diversity of Living Organisms and Viruses. Biophysical Journal, 112(3), 309a. https://doi.org/10.1016/j.bpj.2016.11.1676

‌

Biomedical Science Lab Report

Student’s Name

Institution

Viruses are microscopic living parasitic organisms that are a bit smaller than bacteria. They are unable to live on their own. Therefore, they attach to another living organism, especially like a human, to reproduce and thrive. Predominantly, viruses are known to cause serious medical complications in human life. The black side of the virus is that they easily lead to death. For instance, the outbreak of Ebola in 2014 from West Africa and swine flu of 2009 that widespread throughout the globe. Recently, the outbreak of Corona Virus (Covid19 Virus) in 2019 that have made the whole globe to remain at a standstill is one of the representations of how dangerous virus is in peoples live. The worst part of it, diseases caused by the virus are difficult to treat, and even getting the vaccine for the same is really hectic.

As viruses host themselves in human beings, they teeter themselves in regions regarded as life. On the one hand, the virus occupies the nucleic acids (DNA and RNA) that makeup living organisms. Ideally, the virus will attach to RNA or DNA (Svintradze, 2017). However, the virus has no ideal capacity to read the codes independently in RNA and DNA. Secondly, the virus is very hard to be noticed due to its microscopic nature. However, their impact on people’s life can be seen easily. When it is attacked by a virus, it is very hard to tell the virus unless quality testing and experiment is done (Isel et al., 2016). For biomedics, this is one of the areas they can specialize in the testing of viruses. The procedure includes taking the samples in the laboratory and conducting the necessary experiments.

The aim of this experiment was to identify which viruses were available in the samples. The importance of detecting which viruses are in a sample or in an individual is to know the medication procedures to be initiated. The potential viruses in this sample are Influenza A and Influenza B, Influenza B, and RSV or Influenza A and Influenza B. However, this will not be substantiated without clear experimentation to determine which among these viruses are in the samples.

Methodology and Results

Extraction of PCR Products

In this session, we started by extracting sample gels that will be used in the experiment to investigate the availability of the potential Viruses. Agarose gel containing 2% off w/v to create bands. The bands were allowed to run for 30 minutes. Fourteen wells were provided to each to help in loading PCR products and provided with equal lanes of the ladder as well. Also, we were provided with 25 microliters of PCR to load to each gel. From these, five microliters were required to be added in each sample and a microliter to be used in loading each dye. (0.25% (w/v)).

The procedure of the extraction of PCR Products

50 microliters of 1Xtbe were added in the gel. After loading, the mixture was run through the electrolysis chamber for approximately 30 minutes, with the electricity current maintained at 100 volts. To ensure the results are correct, the marker was maintained a stable point to prevent running to the end of the gel. The process was monitored closely to see the movement of the dye through the gel. When loading, we ensured that every individual group member was set to prevent his gel. Caution was taken to prevent the staring of the gel without the samples of all other group members. After loading the sample gel, we added five microliters of the DNA ladder to almost the middle of the gel.

Extraction of RNA and RT-PCR

The procedure involves equipment and protocols necessary in the purification of Viral RNA. The initial volume used was 50 microliters. We followed all the required steps necessary to generate cDNA from the RNA template. We used 5 micrograms of purified RNA together with random hexamines to help in priming cDNA synthesis.

The procedure was as follows

The solution to be used was thoroughly vortex and briefly centrifuged before using it. We then prepared the priming prefix by referencing the units of measurements presented to us in table 1. To obtain clear results, the priming premix had to be prepared using the ice in the RNase free reaction tube. The reaction was then mixed gently by the use of a pipette. Pipette ensured that the solution thoroughly mixed but without interfering with solution temperature in case there was extreme mixing. After mixing, the solution was kept in incubation at 25 degrees for exactly 10 minutes. Afterward, the solution was put in the same incubation at 45 degrees for 30 minutes. To finish the reaction, the solution was incubated for 85 degrees for 5 minutes then hold at 4 degrees. After this, we stored the solution for future use in the experiment at -20 degrees. This was to ensure that no bacteria or new virus can enter at low temperatures as bacteria survive at normal body temperature.

To complete this experiment, the following primer designs were used.

After obtaining the primers, we conducted a laboratory test to determine whether the named viruses were available in the solution. From the word go, we identified Influenza A virus to be available in the samples. Therefore, the whole experiment was to determine if Influenza A virus was available.

We pipetted 15 microliters of master mix from the previously prepared PCR placed in an MM tube. After producing multiplex PCR, we merged all primers into a single testing tube. We later on added 8 microliters to al tubes containing PCR prime mergers, and we marked them. After completing this procedure, all our tubes had the needed primers dNTPs, Taq, Water, and buffer all equal to 23 microliters. With our cDNA, we had prepared earlier and stored in incubation at -20 degrees we added it to each PCR tube of 23microliters master mix. Plus, the 2 microliters of cDNA, the total volume was 25 microliters.

Sequencing

We used the Hartwell Center of Biotechnology to determine the sequencing of the DNA by the use of oligonucleotides cycles together with AmpliTaq DNA polymerase.

Results and Observation

Genes Targeted in the PCR and the respective primers

Most of Influenza viruses comprises eight vRNA segments with a polarity of negative. The virus has a genome of A/PR/8/34 (H1N1) that is 13,588 nucleotides long. The genome is divided into several sections. Polymerase genome, both PB1, and PB2 are made up of 2341 nucleotides each. A nucleotide has 1565, Matrix gene 1027, nonstructural 890, and HA has 1778 nucleotides. From the primers obtained, the region of passive coding is made of 5 termini with vRNA made of 20-58 long nucleotides (Nadia & Sadok, 2017). The length of 3 lies between the 19^th and 45^th nucleotides. As seen from the sequencing, the difference is existing within these segments, which are classified into two parts. The first classification is made of 13 stored nucleotides located at each 5’end and the other conserved at 12 nucleotides at 3’-end. All these nucleotides are stored in each vRNA to all influenza viruses.

Substitution is a necessary analysis that is done to any primers found in the 14 or 15, which is found in the 3 ends similar to that found in the 21 (5′-end) terminal nucleotide. The PCR temperatures remained to anneal. To control this, we added the 5^th sequence primer of the noninfluenza virus. This is because this design allows primer amplification for different segments as they differ by as little as 2 or three nucleotides at the 3′-end. We, later on, performed the RT-PCR two-step analysis. The first analysis involved a 12 Uni primer, which is a 12 complimentary of the stored nucleotides at the vRNA conserved at the 3-end. The final step was that of PCR, which included a reverse-transcription of products that were amplified using the primers from segment-specific like Protein nucleotides.

Internal gene analysis of RT-PCR

In this analysis, we tried to determine whether the difference between primers could allow amplification of a specific full-length of the corresponding gene. We, therefore, performed RT-PCR, which had specific primers located at each segment (Doris et al., 2017). The results are shown below.

The diagram represents the preserved terminals in the regions of 3 and 5 across all eight segmentations of Influenza A vRNA together with all universal primers. All the segmentations differ in length but have similar characteristics of segmentation. As observed earlier, all segmentation in 13’ vRNA conserved nucleotides has 5 termini to ever influenza virus. For 13 segmentation is made of 5′ terminus. The segmentation has the Oligo-V sequence that is necessary for mRNAs for polyadenylation. The dash region shows regions of non-conformity.

Results for gel Electrophoresis

The full-length amplification if

The figure shows the photo taken from the electron microscope as a representation of the electrolyzed gel. The full-length amplification included the total tubes filled with the gel containing the virus. The electrolyzed PCR was isolated to help and reverse transaction processes performed to collect Uni12 primer. Subsequently, the electrophoresis process was done to all unknown samples, DNA ladder, and even to the positive controls. Almost 10% of the PCR produced was subjected to direct electrophoresis that contained 1% of agarose gel. To make results more clear, we added ethidium bromide.

Discussion and Conclusion

Choosing the right primer sequences is an essential variable to determine the specificity of viruses in the given samples based on the RT-PCR methods. For this report, we tried d to show how segmentation of all samples containing the virus can be amplified using the RT-PCR method by using several primers (Universal). First, all three lines indicated the presence of universal primers at that is seen at the 3′ end. Available viruses could, therefore, be isolated in one of the samples. Secondly, the database indicated a series of segmentation in different terminus that are found in lane 3 and 5. Our primers included NP, PA, PB2, and PB1. All these had G-residue containing M, HA, and NS primers. Third, we conducted mutational analyses. From this analysis, we found that vRNA was being promoted to secondary structure with a confirmation of a double formation to each specific position like in the region 12 and 13.

The complementary of 12 and 13 mer oligonucleotides is well described by Adeyefa and coauthors by the use of the multiplex RT-PCR method. This resulted in the amplification of the eight regions of RNA (Deubel & T, 2017). However, their primers differ from the primers we used in our experiment. Our primers allowed amplification of the segments fully to ensure the potential virus is clearly seen under an electronic microscope. We based our RT-PCR on the 12^th and 13^th conserved nucleotides. However, the cloning of these segments could be impossible to give us the real virus available in the samples. To overcome this drawback, we used primers we developed through electrophoresis and added them to each specific segment, which helped in the amplification of the intended segment. Gel electrophoresis was used to separate the subtypes of PCR like HA and NA. Zou tried to use different sizes of primers in his experiment. However, our experiment was strictly for 3′ and 5′ length of each specific region that contained the sequences of non-influenza virus (Deubel & T, 2017). Therefore, the presence of sequence analysis worked well in our experiment to help identify the right virus in the experiment.

The virus described above has the following characteristics. The virus has eight segmentation with nucleotides ranging from 12 and 13. The length of the strands ranges from 3’ and 5’ in length. Also, we found the available virus to contain nucleoprotein, nonstructural proteins. The primers of the available virus did not work well under cloning. All these characteristics reflect only one class of viruses; the Influenza A virus.

References

Deubel, V., & T, F. (2017). Virus Identification – an overview | ScienceDirect Topics. Www.Sciencedirect.Com. https://www.sciencedirect.com/topics/medicine-and-dentistry/virus-identification

‌ Doris, P. A., Hayward-Lester, A., & Hays, J. K. (2017). Q-RT-PCR: data analysis software for measurement of gene expression by competitive RT-PCR. Bioinformatics, 13(6), 587–591. https://doi.org/10.1093/bioinformatics/13.6.587

‌ Isel, C., Munier, S., & Naffakh, N. (2016). Experimental Approaches to Study Genome Packaging of Influenza A Viruses. Viruses, 8(8), 218. https://doi.org/10.3390/v8080218

‌ Nadia, B., & Sadok, S. (2017). Comparison of DNA-extraction Methods Suitable for PCR-based Applications to Identify Shrimp Species in Commercial Products. Journal of FisheriesSciences.Com, 11(4). https://doi.org/10.21767/1307-234x.1000138

‌ Svintradze, D. V. (2017). Geometric Diversity of Living Organisms and Viruses. Biophysical Journal, 112(3), 309a. https://doi.org/10.1016/j.bpj.2016.11.1676

‌