Taguchi optimisation of a multiplex pneumococcal serotyping PCR and description of 11 novel serotyping primers.
Recently, a PCR-derived method for serotyping Streptococcus pneumoniae has been devised to substitute the conventional antiserum phenotypic method. The method initially used a multiplex PCR reaction, dividing the isolates into 6 different groups based on the detected PCR gel pattern. In order to optimise and refine this crucial step, the Taguchi technique was employed, which can evaluate the individual effect of six parameters (in this case: primers, MgCl2, nucleotide mix, polymerase and buffer), with only 18 experiments; varying the parameter levels in an orthogonal matrix which suppresses the interactions between them. With this method, clear and sharp bands were observed in 5 experiments out of the 18, while the PCR did not work reliably in the remaining cases. In addition, the PCR-based technique could be rendered more economic by the 10-fold lowering of the quantities of two primers. The modified reaction yielded identical results to those obtained with the original method. Furthermore, we have designed serotype-specific primers for 11 new serotypes. The most important ones are those that can distinguish the very closely related, but equally important serotypes 6A and 6B.
Dobay
Orsolya
O
Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary. dobors@net.sote.hu
Juhász
Emese
E
Ungvári
Agnes
A
Jeney
Cs
C
Amyes
S G B
SG
Nagy
K
K
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011008:Pneumococcal Infections / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D012703:Serotyping / Q000379:methods*; D013045:Species Specificity; D013296:Streptococcus pneumoniae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers