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Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Published on: August 6, 2011
Solid-phase PCR with hybridization and time-resolved fluorometry for detection of HLA-B27
M Sjöroos1, J Ilonen, T Lövgren
1JDF Center for Prevention of Type 1 Diabetes in Finland, and Department of Virology, University of Turku, Turku, Finland. minna.sjoroos@utu.fi
Clinical Chemistry
|March 10, 2001
Summary
This study demonstrates a new solid-phase PCR method for detecting HLA-B27 alleles. This approach enables multiple DNA targets to be analyzed efficiently in a single microtiter well.
Area of Science:
- Molecular Biology
- Genetics
Background:
- Preactivated solid surfaces enable multi-step reactions in microtiter plate formats.
- This study focuses on detecting Human Leukocyte Antigen B27 (HLA-B27) alleles.
Purpose of the Study:
- To develop and evaluate a novel solid-phase PCR method for HLA-B27 allele detection.
- To enable multiplex DNA detection and analysis within a single microtiter well.
Main Methods:
- Multiplex solid-phase PCR was performed on a NucleoLink surface to amplify HLA-B27 alleles and beta-actin.
- PCR was followed by hybridization and time-resolved fluorescence detection.
- Covalent immobilization of PCR primers was achieved using amination and a poly(dT)10 linker via carbodiimide chemistry.
Main Results:
- Amination with a poly(dT)10 linker proved superior for stable primer immobilization, with at least 19.5% attachment.
- The novel method demonstrated full agreement with the standard HLA-B27 detection system in 82 genomic DNA samples and dried-blood spots.
- High efficiency and stability of primer immobilization were achieved.
Conclusions:
- The developed solid-phase PCR approach is suitable for multiplex DNA detection.
- This method allows for rapid PCR and hybridization in a few hours using precoated strips and dried-blood spot templates.
- Enables efficient and reliable detection of specific DNA alleles.
