A complete pathogen detection assay based on MIP technology is under construction. Here, we describe a successful model assay targeting 24 HPV genotypes most commonly associated with the progression to cervical cancer. Our pilot subset can now be extended to include all known sequenced HPV genotypes, as multiplexing up to 20,000 using the MIP method for human SNP genotyping, has been demonstrate to be feasible (www.affymetrix.com/technology/mip_technology.affx
), and this would more than cover the existing variability in the HPV genome.
In the case of a double HPV infection, the fact that the PathogenMip Assay detected and distinguished between two contributing viral loads that differed by 2-logs at an input level of 200ng genomic DNA is quite promising. While DNA yields from tumors vary widely, this level is in the mid-range of recovery in our experience. Exploration of a more full matrix of DNA double-loads and inputs is under way.
Primary tumor genomic DNA extracts were efficiently genotyped for a wide dynamic range of viral loads (). The reference probe (rMIP) provided a positive reference control in each performed reaction () and, if used to normalize viral loads against total human genomic DNA content, may allow the researcher a tool to compare viral loads from genomic DNA extracts. The results of the PathogenMip Assay genotyping were in 100% agreement with the conventional genotyping methods.
Our goal was to provide a complete, experimentally validated assay in a ready-to-use format, which could be applied generically to any pathogen based on genome target recognition. PathogenMiper 
serves as an in silico
multiplex test system, whereby one can check the feasibility of designing a MIP assay for the specific targets one might want to include in a multiplex detection assay.
Microbial genomes commonly incur variations from insertions, deletions, or mutations that can generate false-negative results if the variation resides within a probes' targeted region. A more complete diagnostic approach would be to include multiple probes per genotype; this would also provide deeper epidemiological insights in functional genome patterns and variations, i.e. mutations coding for drug resistance 
. Conventional PCR-based genotyping methods are restricted to reside within conserved genomic regions, which limits the choice of genotyping targets 
. These methods are not robust to genomic changes, a problem that can be at least partially addressed by the multiplexing possible in the PathogenMip Assay.
In the current incarnation of the assay, we have incorporated a ligation step to facilitate probe generation. An oligonucleotide complementary to the universal primer region, which is present in each probe, can be used as a bridge so that one longer probe can be ligated together from two smaller constructs, and length requirements for probe synthesis are therefore cut in half. Longer oligonucleotides result in higher initial costs, and have been known to compromise sequence quality 
. With our new ligation-based probe production, we introduce cost-efficient probe production with possibilities for innovation and potential increase in efficiency. Probe production using the ligation method can easily be performed in any academic or other research institution with budget constraints, using only standard labware and avoiding core facility based research. We are currently investigating parameters for increased ligation efficiency and probe production. The goal of such efforts would be a more homogeneous distribution of probe concentrations, thus enabling one to perform high-throughput production via one-tube reactions and use of fully automated workstations, e.g. Biomek FX (Beckman Coulter, Palo Alto, CA). Parameters that could be varied to this end include ligation at higher temperatures (preferred 65°C) to allow improved incubation profiles, and higher cycle numbers 
. Use of modified nucleotides, e.g. locked nucleic acids (LNA) 
in the bridge would allow for higher melting temperatures (Tm
) for the universal primer regions. Also by replacing the conventional gel-purification protocol with one based on single-strand DNA separation, higher purity of ligated versus non-ligated DNA can be achieved, resulting in a more sensitive assay (data not shown). The introduction of a second universal primer region would require a ligation-based production assay based on three contributing constructs and two bridges complementary to the two universal primer regions. By designing the new probes so that the two primer regions flank the barcode region, a three-way ligation currently under investigation would result in new design features regarding assay possibilities and efficiency.
Although our preferred readout for the PathogenMip Assay uses sequencing or hybridization of the identifying barcode, we also used sequencing of the target-homologous regions of the PathogenMip inverted probes to double check target identity. In addition, we performed conventional HPV identification through the sequencing of the L1 regions. All three assays (PathogenMip/barcode, PathogenMip/target region, and HPV L1 sequencing) produced the same final result with respect to target identification, regardless of whether the readout was performed by pyrosequencing or hybridization methods. Pyrosequencing of the ID-tag was more time and cost-efficient than either hybridization to the in-house barcode-chip or the L1-hybridization-assay. Multiple-primer pyrosequencing, however, is limited to reactions of 20-plex or lower. A higher degree of multiplexing in a sequencing-based approach might be achieved through use of a high-throughput configuration of the pyrosequencing method; for example, the one offered by 454 Life Sciences (http://www.454.com/applications/index.asp
). However, when high capacity is required, hybridization-based methods show more potential than sequencing methods, and here the PathogenMip Assay has the advantage of providing more discrimination through its targeted probes than would a standard hybridization to highly conserved regions. The use of ID-tag markers incorporated in the probes simplified sequence read-out in the current PathogenMip Assay, but a more forward looking approach involves refinement of the in-house barcode-chip hybridization piloted here. We anticipate that a high-throughput configuration of the current PathogenMip assay will be achievable through use of the commercially available Affymetrix TAG4-array 
, which is based on detection of barcodes through hybridization similar to the process described here. The TAG4-array covers a total of 80K features, which includes a total of 16,000 unique barcodes in five replicates.
Sensitivity levels obtained with the current assay were sufficient for a functional assay and were improved with the use of a more extensive amplification protocol. Previously described ligation-based genotyping methods 
have shown sensitivity ranging down to nanograms of genomic DNA for accurate genotyping. These methods have placed careful emphasis on the amplification step by either rolling circle amplification method 
or stringent reaction cleanup with reversible magnetic biotin-streptavidin separation 
, followed by design of a PCR run at high annealing temperatures and high cycle numbers, which also could be performed with the use of LNA universal primers 
. Further consideration of these parameters should allow improvement in sensitivity of a next-generation PathogenMip Assay.
The potential uses for an assay that simultaneously detects multiple organisms based on differences in DNA sequences are almost unlimited. MIP-based diagnostics have already progressed in the field of human SNP-detection and cancer diagnostics 
with ongoing clinical trials. Microarray techniques have gained ground in the pathogen diagnostic arena, with various genotyping chips such as the HPV-assay PapilloCheck (www.greinerbioone.com
) upcoming. By combining the benefits of a high-throughput multiplex MIP-assay prior to PCR amplification and of a post-PCR hybridization based chip read-out, researchers can expand the scope of their surveillance to reach new insights into epidemiology and clinical management. We therefore encourage researchers with expertise in various pathogenic microbes to adapt the PathogenMip Assay to accomplish similar detection screenings as performed in this study. By combining the different models, we can reach a common goal toward a standardized virus-, bacteria-, or fungi- chip for routine diagnostics and epidemiological studies.