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PMID: 17311101 Published · epublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Validation Study

PathogenMip assay: a multiplex pathogen detection assay.

PloS one ·Vol. 2 ·No. 2 ·2007-02-21 ·Pages e223

Akhras MS, Thiyagarajan S, Villablanca AC, Davis RW, Nyrén P, Pourmand N

Abstract

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.

MeSH Terms
Alphapapillomavirus/genetics,isolation & purification DNA Ligases DNA Probes, HPV/chemical synthesis,isolation & purification DNA, Neoplasm/analysis DNA, Viral/analysis Disease Progression Electronic Data Processing Female Genotype Humans Oligonucleotide Array Sequence Analysis Papillomavirus Infections/diagnosis,pathology,virology Polymerase Chain Reaction/methods Sensitivity and Specificity Sequence Analysis, DNA Uterine Cervical Neoplasms/pathology,virology
Chemicals
DNA Probes, HPV DNA, Neoplasm DNA, Viral DNA Ligases
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Akhras Michael S
Stanford Genome Technology Center, Stanford University, Palo Alto, California, United States of America.
Thiyagarajan Sreedevi
Villablanca Andrea C
Davis Ronald W
Nyrén Pål
Pourmand Nader
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Article Info
Journal
PloS one
Abbr.
PLoS One
ISSN
1932-6203
DOI
10.1371/journal.pone.0000223
Published
2007-02-21
Epub
2007-00-21
Pages
e223
Language
English
Region
United States
NLM ID
101285081
PMCID
PMC1794193
Subset
IM
Grants
NHGRI NIH HHS · P01 HG000205 · United States
NHGRI NIH HHS · (PO1-HG000205) · United States
External Links
PubMed source DOI: 10.1371/journal.pone.0000223 PMC Full Text View journal details
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