Limits...
The application of multiplex PCR to detect seven different DNA targets in group B streptococci.

Gosiewski T, Brzychczy-Włoch M, Heczko PB - Folia Microbiol. (Praha) (2012)

Bottom Line: GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains.Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10).Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection.

View Article: PubMed Central - PubMed

Affiliation: Jagiellonian University Medical College, Cracow, Poland. tomasz.gosiewski@uj.edu.pl

ABSTRACT
Group B Streptococcus (GBS) causes severe infections in infants and in immunocompromised adults. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. For this reason, it is important to be able to carry out immediate and comprehensive diagnostics of these infections. Seven genes important for screening of GBS infection were detected: cfb gene encoding the CAMP factor presented in every GBS; the cps operon genes such as cps1aH, cps1a/2/3IJ, and cps5O specific for capsular polysaccharide types Ia, III, and V, respectively; macrolide resistance genes ermB and mefA/E; and the gbs2018 S10 region specific for ST17 hypervirulent clone. Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10). Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection.

Show MeSH

Related in: MedlinePlus

Results of standardization of the multiplex PCR for detection of seven different sequences important for screening of group B Streptococcus infection (1—hypervirulent clone ST17; 2—12403 ATCC, III serotype; 3—BAA611 ATCC, V serotype; 4—strain with mef A/E gene; 5—strain with ermB gene; 6—11360 NCTC; Ia serotype; 7—the negative control)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3345335&req=5

Fig1: Results of standardization of the multiplex PCR for detection of seven different sequences important for screening of group B Streptococcus infection (1—hypervirulent clone ST17; 2—12403 ATCC, III serotype; 3—BAA611 ATCC, V serotype; 4—strain with mef A/E gene; 5—strain with ermB gene; 6—11360 NCTC; Ia serotype; 7—the negative control)

Mentions: The total volume of the designed multiplex PCR reaction was 40 μL and it consisted of: seven pairs of primers (concentration presented in Table 1); 50 ng of bacterial DNA; 250 μmol/L of each dNTP (Fermentas); 1.87 mmol/L MgCl2, 2 U of Taq polymerase (EURx); and KCl buffet for polymerase (EURx). The developed program for multiplex PCR amplification was devised using the following thermal profile: 95°C—5 min, 50 × (95°C—1 min, ramp 0.3°C to 52°C, 52°C—1 min, 72°C—3 min), 72°C—10 min (PTC-200, BioRad). The efficacy of the multiplex PCR method was tested with the application of the group of reference strains with confirmed presence of the desired genetic sequences (Fig. 1).Fig. 1


The application of multiplex PCR to detect seven different DNA targets in group B streptococci.

Gosiewski T, Brzychczy-Włoch M, Heczko PB - Folia Microbiol. (Praha) (2012)

Results of standardization of the multiplex PCR for detection of seven different sequences important for screening of group B Streptococcus infection (1—hypervirulent clone ST17; 2—12403 ATCC, III serotype; 3—BAA611 ATCC, V serotype; 4—strain with mef A/E gene; 5—strain with ermB gene; 6—11360 NCTC; Ia serotype; 7—the negative control)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3345335&req=5

Fig1: Results of standardization of the multiplex PCR for detection of seven different sequences important for screening of group B Streptococcus infection (1—hypervirulent clone ST17; 2—12403 ATCC, III serotype; 3—BAA611 ATCC, V serotype; 4—strain with mef A/E gene; 5—strain with ermB gene; 6—11360 NCTC; Ia serotype; 7—the negative control)
Mentions: The total volume of the designed multiplex PCR reaction was 40 μL and it consisted of: seven pairs of primers (concentration presented in Table 1); 50 ng of bacterial DNA; 250 μmol/L of each dNTP (Fermentas); 1.87 mmol/L MgCl2, 2 U of Taq polymerase (EURx); and KCl buffet for polymerase (EURx). The developed program for multiplex PCR amplification was devised using the following thermal profile: 95°C—5 min, 50 × (95°C—1 min, ramp 0.3°C to 52°C, 52°C—1 min, 72°C—3 min), 72°C—10 min (PTC-200, BioRad). The efficacy of the multiplex PCR method was tested with the application of the group of reference strains with confirmed presence of the desired genetic sequences (Fig. 1).Fig. 1

Bottom Line: GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains.Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10).Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection.

View Article: PubMed Central - PubMed

Affiliation: Jagiellonian University Medical College, Cracow, Poland. tomasz.gosiewski@uj.edu.pl

ABSTRACT
Group B Streptococcus (GBS) causes severe infections in infants and in immunocompromised adults. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. For this reason, it is important to be able to carry out immediate and comprehensive diagnostics of these infections. Seven genes important for screening of GBS infection were detected: cfb gene encoding the CAMP factor presented in every GBS; the cps operon genes such as cps1aH, cps1a/2/3IJ, and cps5O specific for capsular polysaccharide types Ia, III, and V, respectively; macrolide resistance genes ermB and mefA/E; and the gbs2018 S10 region specific for ST17 hypervirulent clone. Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10). Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection.

Show MeSH
Related in: MedlinePlus