Journal List > J Bacteriol Virol > v.38(4) > 1033913

Jang, Yoon, Oh, Lee, and Lee: Development of UPSR Primer Design Program for Efficient Detection of Viruses


PCR is a rapid and sensitive method for detection of viruses from clinical samples and good primers are essential for successful PCR. However, high mutation rate of viral genomes often results in failure in detecting viruses, and there have been attempts to develop primers from multiple viral sequences. Thus, we developed a program called Universal Primers Score Ranking (UPSR) which generates primers from multiple sequences and ranks the quality of primers automatically. The feasibility of the UPSR program was tested using hepatitis B viruses (HBV) isolated from Korean patients. UPSR generated primer candidates with quality score ranks according to two Tm values. We found that Tm2 values calculated based on the thermodynamics of nearest neighboring bases were better correlated with actual detection rate of HBV from patients' sera. The primer with number 1 rank by Tm2 values detected more samples than any other primers designed by UPSR, commercial primer, or other reference primers suggested by previous literatures. Thus, UPSR proved to be easy and useful to design primers from multiples sequences in detecting viruses.


1). Breslauer KJ., Frank R., Blocker H., Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA. 83:3746–3750. 1986.
2). Chen SH., Lin CY., Cho CS., Lo CZ., Hsiung CA. Primer Design Assistant (PDA): A web-based primer design tool. Nucleic Acids Res. 31:3751–3754. 2003.
3). Grant PR., Busch MP. Nucleic acid amplification technology methods used in blood donor screening. Transfus Med. 12:229–242. 2002.
4). Haas S., Vingron M., Poustka A., Wiemann S. Primer design for large scale sequencing. Nucleic Acids Res. 26:3006–3012. 1998.
5). Hamasaki K., Nakata K., Nagayama Y., Ohtsuru A., Daikoku M., Taniguchi K., Tsutsumi T., Sato Y., Kato Y., Nagataki S. Changes in the prevalence of HBeAg-negative mutant hepatitis B virus during the course of chronic hepatitis B. Hepatology. 20:8–14. 1994.
6). Huang YC., Chang CF., Chan CH., Yeh TJ., Chang YC., Chen CC., Kao CY. Integrated minimum-set primers and unique probe design algorithms for differential detection on symptom-related pathogens. Bioinformatics. 21:4330–4337. 2005.
7). Jarman SN. Amplicon: software for designing PCR primers on aligned DNA sequences. Bioinformatics. 20:1644–1645. 2004.
8). Koressaar T., Remm M. Enhancements and modifications of primer design program Primer3. Bioinformatics. 23:1289–1291. 2007.
9). Li P., Kupfer KC., Davies CJ., Burbee D., Evans GA., Garner HR. PRIMO: a primer design program that applies base quality statistics for automated large-scale DNA sequencing. Genomics. 40:476–485. 1997.
10). Nainan OV., Cromeans TL., Margolis HS. Sequence-specific, single-primer amplification and detection of PCR products for identification of hepatitis viruses. J Virol Methods. 61:127–134. 1996.
11). Proutski V., Holmes EC. Primer Master: a new program for the design and analysis of PCR primers. Comput Appl Biosci. 12:253–255. 1996.
12). Raddatz G., Dehio M., Meyer TF., Dehio C. PrimeArray: genome-scale primer design for DNA-microarray construction. Bioinformatics. 17:98–99. 2001.
13). Roth WK., Weber M., Petersen D., Drosten C., Buhr S., Sireis W., Weichert W., Hedges D., Seifried E. NAT for HBV and anti-HBc testing increase blood safety. Transfusion. 42:869–875. 2002.
14). Rozen S., Skaletsky HJ. Primer3 on the WWW for general users and for biologist programmers. pp. p. 365–386. In. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Misener S, Krawetz SA, editors. (Ed),. Humana Press;Totowa, NJ: 2000.
15). SantaLucia J Jr., Allawi HT., Seneviratne PA. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry. 35:3555–3562. 1996.
16). Sugimoto N., Nakano S., Yoneyama M., Honda K. Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res. 24:4501–4505. 1996.
17). Untergasser A., Nijveen H., Rao X., Bisseling T., Geurts R., Leunissen JA. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 35:W71–74. 2007.
18). Wang D., Urisman A., Liu YT., Springer M., Ksiazek TG., Erdman DD., Mardis ER., Hickenbotham M., Magrini V., Eldred J., Latreille JP., Wilson RK., Ganem D., DeRisi JL. Viral discovery and sequence recovery using DNA microarrays. PLoS Biol. 1:E2. 2003.
19). Zaaijer HL., ter Borg F., Cuypers HT., Hermus MC., Lelie PN. Comparison of methods for detection of hepatitis B virus DNA. J Clin Microbiol. 32:2088–2091. 1994.

Figure 1.
Screen capture of the UPSR program. The captured UPSR screen shot shows the quality ranks and scores for primers designed by UPSR.
Figure 2.
Relation between the primer quality ranks and HBV detection rate. Primers with high quality scores according to Tm1 (circle) or Tm2 (triangle) were selected and applied to HBV detection by PCR from 14 HBV sAg(+) serum samples.
Table 1.
Programming steps to develop UPSR program
Steps Programming Name of files generated
1 Generation of consensus sequences in forward and reverse directions 1_Common_forward 2_Common_reverse
2 Selection of sequences with longer than 20 bases 3_base20_forward 4_base20_reverse
3 Examination of degeneracy, Tm, GC%, 3′ Clamp, 5′ GC content, and 3′ GC content 5_shift_forward 6_shift_reverse
4 Selection of the primer candidates 7_Universal
5 Calculation of the Tm values 8_Tm
6 Examination of the hairpin generation 9_hairpin
7 Examination of the dimer generation 10_dimer
8 Calculation the quality measures and rank the scores according to Tm1, Tm2 11_ranking
9 Sorting the primer quality scores based on Tm1 12_sort_1
10 Sorting the primer quality scores based on Tm2 13_sort_2
Table 2.
Primers designed and tested in this study
Tm Rank Quality Score Prime sequencea (F/R, 5′-3′) Locationb Size Primer name
  1 104.66 CCCAACCTCCAATCACTCAC 319 159 CB-1
  4 102.87 ACTCGTGGTGGACTTCTCTC 252 226 CB-2
Tm1 10 101.46 CCAACCTCCAATCACTCACC 320 158 CB-3
  14 100.83 GACTCGTGGTGGACTTCTCT 251 227 CB-4
  20 98.36 ACCTCCAATCACTCACCAAC 323 155 CB-5
  1 105.30 CCCAACCTCCAATCACTCAC 319 160 CB-6
  4 102.16 CCCAACCTCCAATCACTCAC 319 159 CB-1
Tm2 10 99.36 CTCCAATCACTCACCAACCT 325 154 CB-7
  14 98.16 ACCTCCAATCACTCACCAAC 323 156 CB-8
  20 94.63 GACTCGTGGTGGACTTCTCT 251 227 CB-4

a Primer sequences are presented in 5′ to 3′ direction. F, forward primer; R, reverse primer

b Location of the primer according to the full genomic sequence of HBV reference strain NC_003977

Table 3.
Detection of HBV by PCR using primers with different quality ranks designed by UPSR program
Primer quality rank Percent detected (± SEM)
Tm1 Tm2
1 71±6 88±4
4 61±9 70±4
10 86±0 64±6
14 46±4 54±4
20 64±0 23±4

Data are averages of four independent experiments with standard errors of the mean (SEM)

Table 4.
Detection of HBV from 38 Korean HBV sAg(+) serum samples by PCR using CB-6 and reference primers
Primer Detection rate Source
CB-6 29 (76%) This study
Commercial 19 (50%) Company “B”
409.703A 18 (45%) Reference 10
686.457 17 (45%) Reference 10
Hamasaki 16 (42%) Reference 5
Similar articles