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Following the optimization of primer set concentrations in multiplex PCR assays (Tables 1 and 2), the specificity of each primer set was also evaluated. For two protocols of the multiplex PCR, each corresponding DNA and combinations of all DNA were amplified successfully without non-specific bands (Figure 1). Each PCR product could be observed as a clear band at 1,103, 600, 217, 504, 356, 500, and 345 bp generated by uidA, lacZ, gyrB, int, invA, and ompW primer sets, respectively. In addition to the above-mentioned bacteria, genomic DNA was also extracted from reference strains (listed in Table 3) and used as a template in the PCR assays. Lack of amplifications for these bacterial strains confirms the high specificity of the newly designed primer sets.
Table 3

Specificity of the primer sets

 PCR results
Reference strainsECShSaVP
Enterococcus faecalis ATCC 29212 (PTCC 1778) − − − − − − 
Enterococcus faecalis ATCC 19433 (PTCC 1774) − − − − − − 
Proteus vulgaris PTCC 1079 − − − − − − 
Pseudomonas putida ATCC 12633 (PTCC 1694) − − − − − − 
Serratia marcescens ATCC 13880 (PTCC 1621) − − − − − − 
Staphylococcus aureus ATCC 25923 (PTCC 1431) − − − − − − 
Streptococcus pyogenes ATCC 19615 (PTCC 1762) − − − − − − 
Bacillus cereus ATCC 11778 (PTCC 1015) − − − − − − 
Salmonella typhi PTCC 1609 − − − − − 
Enterobacter aerogenes ATCC 13048 (PTCC 1221) − − − − − 
E. coli NCIMB 11032 (PTCC 1395) − − − − 
Vibrio cholerae PTCC 1611 − − − − − 
 PCR results
Reference strainsECShSaVP
Enterococcus faecalis ATCC 29212 (PTCC 1778) − − − − − − 
Enterococcus faecalis ATCC 19433 (PTCC 1774) − − − − − − 
Proteus vulgaris PTCC 1079 − − − − − − 
Pseudomonas putida ATCC 12633 (PTCC 1694) − − − − − − 
Serratia marcescens ATCC 13880 (PTCC 1621) − − − − − − 
Staphylococcus aureus ATCC 25923 (PTCC 1431) − − − − − − 
Streptococcus pyogenes ATCC 19615 (PTCC 1762) − − − − − − 
Bacillus cereus ATCC 11778 (PTCC 1015) − − − − − − 
Salmonella typhi PTCC 1609 − − − − − 
Enterobacter aerogenes ATCC 13048 (PTCC 1221) − − − − − 
E. coli NCIMB 11032 (PTCC 1395) − − − − 
Vibrio cholerae PTCC 1611 − − − − − 

PTCC: Persian Type Culture Collection.

E: E. coli-specific primers.

C: Coliform-specific primers.

Sh: Shigella spp.-specific primers.

Sa: Salmonella spp.-specific primers.

V: Vibrio cholerae-specific primers.

P: Pseudomonas aeruginosa-specific primers.

+, Positive PCR result.

−, Negative PCR result.

Figure 1

Gel agarose analysis of uniplex and multiplex PCR protocols of spiked water. Lane 1, Enterococcus faecalis (ATCC 29212) as negative control; Lane 2, Pseudomonas putida (ATCC 12633) as negative control; Lane 3, gyrB for Pseudomonas aeruginosa (ATCC 27853) (217 bp); Lane 4, int for Shigella sonnei (ATCC 9290) (356 bp); Lane 5, uidA for E. coli (ATCC 25922) (1103 bp); Lane 6, triplex for gyrB, int, and uidA; Lane 7, DNA 100 bp ruler (Thermo Scientific); Lane 8, lacZ for E. coli (ATCC 25922) (600 bp); Lane 9, invA for Salmonella typhimurium (ATCC 14028) (500 bp); Lane 10, ompW for Vibrio cholera (ATCC 14035) (345 bp), and Lane 11, triplex for invA, ompW, and lacZ; Lane 12, blank (nothing loaded).

Figure 1

Gel agarose analysis of uniplex and multiplex PCR protocols of spiked water. Lane 1, Enterococcus faecalis (ATCC 29212) as negative control; Lane 2, Pseudomonas putida (ATCC 12633) as negative control; Lane 3, gyrB for Pseudomonas aeruginosa (ATCC 27853) (217 bp); Lane 4, int for Shigella sonnei (ATCC 9290) (356 bp); Lane 5, uidA for E. coli (ATCC 25922) (1103 bp); Lane 6, triplex for gyrB, int, and uidA; Lane 7, DNA 100 bp ruler (Thermo Scientific); Lane 8, lacZ for E. coli (ATCC 25922) (600 bp); Lane 9, invA for Salmonella typhimurium (ATCC 14028) (500 bp); Lane 10, ompW for Vibrio cholera (ATCC 14035) (345 bp), and Lane 11, triplex for invA, ompW, and lacZ; Lane 12, blank (nothing loaded).

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