Design multiplex PCR primer and probe gen ORF1ab and gen N on COVID-19

INTRODUCTION

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS virus CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) which is a new strain of Coronavirus.¹ On July 11, 2021, the Government of the Republic of Indonesia reported as many as 2,527,203 positive cases with deaths reaching 66,464 people, COVID-19 cases continue to increase and are difficult to control. COVID-19 spread to different countries of the world and became public healtthreatts globally in a relatively short period.²

Over time, the coronavirus will undergo genetic mutations. Gene mutation is a spontaneous genetic change from a parent virus particle to a derivative viral particle. The coronavirus gene is composed of a series of ribonucleic acid (RNA), the genetic sequence in the coronavirus is the genome of the coronavirus.³ The genome in SARS-CoV-2 consists of a single strand of RNA with a size of 29,903 base pairs, two-thirds of the viral genome sequence is a non-structural protein encoder in the ORF1ab gene that plays a role in RNA synthesis by amplifying the target gene in SARS-CoV-2.⁴ The virulence mechanism of the coronavirus is related to structural proteins and non-structural proteins. Coronavirus provides messenger RNA (mRNA) that can help the translation process of the replication/ transcription process. The genes that play a role in this replication and transcription process include two-thirds of the 5'-end RNA sequence and two overlapping Open Reading Frames (ORFs), namely ORF1a and ORF1b.⁵ There are 16 non-structural proteins encoded by ORF. The other 1/3 part of the viral RNA circuit, which does not play a role in the replication/transcription process, plays a role in encoding 4 structural proteins, namely the S protein (spike), protein E (envelope), protein M (membrane), and protein N (nucleocapsid).⁶

The N gene is a structural protein that binds to the virus’s genetic material, encodes the formation of nucleocapsid protein, and is a conserved region, so there is very little chance of it mutating.⁷ The N gene is one of the targets that have a very high analytical sensitivity, making the N gene the most sensitive polymerase chain reaction (PCR) diagnostic test target recommended by the CDC, United States.⁸ Along with the development of molecular biotechnology, bioinformatics which is a computational technique for managing and analyzing biological information has developed rapidly. In this study, by utilizing bioinformatics, the design of primers and probes with the multiplex PCR method on the ORF1ab gene and N genes is expected to be able to become good validity if there are certain mutations or variants in SARS-CoV-2 in the future, that it can be used as a specific primary candidate and probe in the detection of COVID-19.

METHODS

The research design used is descriptive research in the form of cross-sectional studies. The population is a sequence of the ORF1ab gene and the N gene with a total sample of 221 COVID-19 gene sequences downloaded from the NCBI and GISAID gene databases. The sampling technique used is in the form of non-random purposive sampling. The research was conducted at the Bioinformatics Research Laboratory of the Faculty of Medicine, University. Data analysis of the results of the study was carried out descriptively.

RESULTS

Primary and probe design analysis on ORF1ab and N genes. The design of primers and probes was carried out to search for the best primary candidates and probes to be used for COVID-19 detection using the multiplex PCR method. Primers and probes were designed using the CLC Genomic Workbench 21 program, a total of 221 sequences in the ORF1ab gene and N genes collected from several countries in the NCBI and GISAID database downloads can be seen in Table 1. The ORF1ab gene is at 266 to 21,555 nucleotide bases. encoded more than 16 non-structural proteins, primers, and orf1ab gene probes designed to measure 183 bp, on specific forward primers amplifying from sequences 15601 to 15619, reverse primers 15765 to 15783 and probes in sequences 15717 to 15737 as well as specific attachment sites to primary attachment sites and ORF1ab gene probes on SARS-CoV-2 RNA can be seen in Figure 1.

The N gene is at 28,274 to 29,533 nucleotide bases, the N gene primer and probe are designed to measure 111 bp, the forward primer on the specific gene amplifies from the order 28858 to 28875, the reverse primer 28950 to 28968 and the specific probe amplifies from sequence 28921 to 28939. The primary attachment site and N gene probe on SARS-CoV-2 RNA can be seen in Figure 2.

DISCUSSION

The design of primers and probes was carried out to search for the best primary candidates and probes to be used for COVID-19 detection using the multiplex PCR method. Sequence alignment is performed to arrange DNA, RNA, or protein sequences to identify areas of similarity that may be a consequence of functional, structural, or evolutionary relationships between sequences. Alignment is carried out using sequence reference (NC_045512.2) sequences are molecularly analyzed to determine the conserved region which is a region free of mutations so that amplification can be carried out.⁹ The essential quality of the ORF1ab quality, the N quality assembly the prerequisites for qRT-PCR can be seen in Table 2. The essential match within the ORF1ab quality brought about in an amplicon estimate of 183 bp and an N quality of 111 bp. The ideal measure for PCR items is 50- 150 bp.¹⁰ In any case, PCR items measuring up to 400 bp can moreover be well intensified.¹¹ so PCR products along 183 bp within the ORF1ab quality are still well opened up. The primary sequence in the ORF1ab gene, the N gene has a length of 18 bases on the forward and reverse, and the probe sequence in the ORF1ab gene has a length of 21 bases and the N gene is 19 bases. The length of the primer and probe meets the requirements in the design of the primer and probe to be used as detection in PCR The primary length meets the requirement of between 18 to 30 bases of the number of bases in the primary sequence and probe. Determines the efficiency of the PCR process.¹² Primary composition is one of the important considerations because it can reduce primary specificity, in the form of GC percentages of the amount of guanine and cytosine in a primer.¹³

The high presentation of the GC will encourage the primer to form a dimer, affecting the amplification results.¹⁴ It can be seen in table 1. Primers designed on the ORF1ab gene have a GC percentage of 42% for primary forward and 42% for primary reverse and in gen N 44% primary forward and 47% primary reverse. Probes designed on the ORF1ab gene have a GC percentage of 43% and in the N gene of 52%, the design of primers and probes with a low GC presentation can reduce the efficiency of the PCR process caused because the primer is unable to compete to attach effectively to the template.¹⁵ Melting temperatures in the primary orf1ab gene and N gene have a Tm that corresponds to the optimal Tm characteristics of 52-65 ºC. In the primary Tm of the ORF 1ab gene with a forward of 54.70 ºC and reverse of 54.16 ºC, the Tm primer of the N forward gene is 55.33 ºC and reverse of 55.09 ºC. The Tm from the probe design results in the ORF1ab gene and the N gene has a Tm that corresponds to the characteristics of 5-10 ºC above the primary Tm, the Tm probe of the ORF1ab gene of 59 ºC, and the N gene of 60 ºC, can be seen in Table 2. The difference in the Tm value of the probe with the primer will ensure that the probe can be hybridized with the target during the primary extension process, thereby increasing the fluorescent signal in the amplification process, a stable hybridization complex will produce maximum exonuclease activity during the PCR process.¹⁶

CONCLUSION

Sequences of primer and probe pairs that can be used for COVID-19 detection using the multiplex PCR method. The results of the primary design of the ORF1ab gene and probe with forward primer: 5'-CGCAATTTACAACACAGAC -3' reverse primer: 5'-GTTCTTTATGCTAGCCACT -3' amplicon length 183 bp, and probe sequence: FAM 5'-AAACACACAACAACCATCGTCA-3' BHQ-1, in N gene with forward primer: 5'-AATTCAACTCCAGGCAG -3' and reverse primer: 5'-CTCTCTCAAGCTGGTTCAATC -3' amplicon length 111 bp, and probe sequence: HEX 5'-CAGCAAAGCAAGAGCAGCA -3' BHQ-1 primer pair sequence and gene probe ORF1ab and the suspected N gene were eligible for q-PCR.

ACKNOWLEDGEMENTS

The author is grateful to the Laboratory of the Center for Diagnostic and Research of Infectious Diseases (PDRPI) Faculty of Medicine of Andalas University, Bioinformatics Research Laboratory of the Faculty of Medicine of Andalas University, Biomedical Laboratory of the Faculty of Medicine of Andalas University. The author would like to thank the Head of the Laboratory of the Center for Diagnostic and Research of Infectious Diseases (PDRPI) Dr. Andani Eka Putra, M.Sc, and other analysts. The authors also would like to thank dr. Rauza Sukma Rita, Ph.D. as a Lecturer at the Department of Biochemistry, Faculty of Medicine, Andalas University, Padang, Indonesia and expresses their gratitude to Juane Plantika Menra, M.Si, Dr. Dede Rahman Agustian as a team that helped with the research.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Institutional Ethics Committee

REFERENCES

1. 1. Li Geng, M Peng, Y Meng, L Lu. Molecular immune pathogenesis and diagnosis of Covid-19. J Pharmaceutical Analysis. 2020;10(2):102-8.
2. 2. Huang CY, Xingwang W, Lili L, Jianping L, Yi ZH. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet. 2019;395(10223):78-9.
3. 3. Khailany RA, Safdar M, Ozaslan M. Genomic characterization of a novel SARS-CoV-2. Gene Rep. 2020;19:100682.
4. 4. Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, Tan W. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA. 2020;323(18):1843-4.
5. 5. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for a polymerase chain reaction. BMC Bioinformatics. 2012;13(134):56-9.
6. 6. Gennaro F, Pizzol D, Marotta C, Antunes M, Racalbuto V, Veronese N, et al. Coronavirus diseases (COVID-19) current status and future perspectives: A narrative review. Int J Environ Res Public Health. 2020;17(8):76-9.
7. 7. Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Müller MA, Wendtner C. Virological assessment of hospitalized patients with COVID-2019. Nature. 2020;581(7809):465-9.
8. 8. Holshue ML, DeBolt C, Lindquist S, Lofy KH, Wiesman J, Bruce H, Pillai SK. The first case of the 2019 novel coronavirus in the United States. New England J Med. 2020;8:45-8.
9. 9. Widowati EW. Primary Design of Cytochrome B (Cyt b) As One of the PCR (Polymerase Chain Reaction) Components for Pig DNA Detection. Lab. Individu Research. 2013: 1-64.
10. 10. Debode F, Marien A, Janssen É, Bragard C, Berben G. The influence of amplicon length on real-time PCR results Frédéric. 2017;21(1):3-11.
11. 11. Holm WV, Ghesquière J, Boon N, Verspecht T, Bernaerts K, Zayed N. A viability quantitative pcr dilemma: are longer amplicons better? Applied Environmental Microbiology. 2019;87(5):1-11.
12. 12. Hung J, Weng Z. Designing polymerase chain reaction primers using primer plus. Cold Spring Harbor Laboratory Press. 2016;7:821-6.
13. 13. Dewi DA, Wardhana AH, Ekawasti F, Sawitri DH. Comparison of five sets of primers for detecting Trypanosoma evansi in mice using the Polymerase Chain Reaction (PCR) In Prosiding National Seminar on Animal Husbandry and Veterinary Technology. 2022;9:178-88.
14. 14. Saraswati H, Seprianto S, Wahyuni FW. Desain Primer Secara In Silico untuk Amplifikasi Gen cryIII dari Bacillus thuringiensis Isolat Lokal. Indonesian Journal of Biotechnology and Biodiversity. 2019;3(1):33-8.
15. 15. Popp J, Bauer M. Modern Techniques for Pathogen Detection. 2015;3:60-2.
16. 16. Bishop JL, Campbell SA, Farrell P, Fitzgerald M, Haugen M, Kocmond W. Designing real-time assays on the smart cycler II system, Cepheid. 2017;8:45-8.