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

Abstract

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.

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Figure 1.
Screen capture of the UPSR program. The captured UPSR screen shot shows the quality ranks and scores for primers designed by UPSR.
jbv-38-259f1.tif
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.
jbv-38-259f2.tif
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
      GGACAAACGGGCAACATACC 477    
  4 102.87 ACTCGTGGTGGACTTCTCTC 252 226 CB-2
      GGACAAACGGGCAACATACC 477    
Tm1 10 101.46 CCAACCTCCAATCACTCACC 320 158 CB-3
      GGACAAACGGGCAACATACC 477    
  14 100.83 GACTCGTGGTGGACTTCTCT 251 227 CB-4
      GGACAAACGGGCAACATACC 477    
  20 98.36 ACCTCCAATCACTCACCAAC 323 155 CB-5
      GGACAAACGGGCAACATACC 477    
  1 105.30 CCCAACCTCCAATCACTCAC 319 160 CB-6
      AGGACAAACGGGCAACATAC 478    
  4 102.16 CCCAACCTCCAATCACTCAC 319 159 CB-1
      GGACAAACGGGCAACATACC 477    
Tm2 10 99.36 CTCCAATCACTCACCAACCT 325 154 CB-7
      AGGACAAACGGGCAACATAC 478    
  14 98.16 ACCTCCAATCACTCACCAAC 323 156 CB-8
      AGGACAAACGGGCAACATAC 478    
  20 94.63 GACTCGTGGTGGACTTCTCT 251 227 CB-4
      GGACAAACGGGCAACATACC 477    

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
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