Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens.
In this study we describe a novel protocol for rapid molecular analysis of patient samples using a combination of real-time polymerase chain reaction (PCR) and Sanger sequencing. This would normally take 2 working days in the diagnostic laboratory, but using this protocol the process can be completed within 3 h using equipment normally found in the laboratory. The innovative steps in this protocol are the sequencing of the product generated in the diagnostic real-time PCR, addition of a sequencing tail to the PCR primer, which increases the quality of the sequence without loss of sensitivity or specificity, and optimization of the hands-on and instrument steps using modern reagents. The read length of the sequencing step is routinely 250 nucleotides, which is substantially longer than existing rapid sequencing methods, increasing the chances of covering several genetic markers within 1 analysis. As proof of the concept, we used the detection and genotyping of the intestinal parasite Giardia lamblia, but the protocol can be applied to any PCR and sequence-based analysis.
Centre for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, Solna, Sweden.
Tara
T
Wahab
Johan
J
Ankarklev
Marianne
M
Lebbad
Steve
S
Glavas
Staffan
S
Svärd
Daniel
D
Palm
D019411:Clinical Laboratory Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
D017931:DNA Primers