PathogenMip assay: a multiplex pathogen detection assay.
The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.
Akhras
Michael S
MS
Stanford Genome Technology Center, Stanford University, Palo Alto, California, United States of America.
Thiyagarajan
Sreedevi
S
Villablanca
Andrea C
AC
Davis
Ronald W
RW
Nyrén
Pål
P
Pourmand
Nader
N
D052685:Alphapapillomavirus / Q000235:genetics / Q000302:isolation & purification*; D011088:DNA Ligases; D015344:DNA Probes, HPV / Q000138:chemical synthesis / Q000302:isolation & purification; D004273:DNA, Neoplasm / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D018450:Disease Progression; D001330:Electronic Data Processing; D005260:Female; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000473:pathology / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*
D015344:DNA Probes, HPV; D004273:DNA, Neoplasm; D004279:DNA, Viral; D011088:DNA Ligases