Korean J Blood Transfus.  2018 Apr;29(1):33-40. 10.17945/kjbt.2018.29.1.33.

Construction and Application of Internal Control for Laboratory-Developed HTLV PCR

• Kang J
• Shin SM
• Kang JW
• Seo YI
• Min H
• Huh K

• KMID: 2435709
• DOI: http://doi.org/10.17945/kjbt.2018.29.1.33

Abstract

BACKGROUND
For donor samples showing reactive results in a human T-cell lymphotropic virus (HTLV) antibody test along with indeterminate results in Western blot assay, HTLV nucleic acid amplification test using laboratory-developed polymerase chain reaction (PCR) was performed. It is necessary to construct an adequate internal control (IC) to evaluate the accuracy of the results since we did not use an IC in the laboratory-developed PCR.
METHODS
As a competitive IC, plasmid DNA containing the primer recognition sequence for amplification of the HTLV pX region was constructed. We determined the adequate concentration of the IC, which was added to the samples to evaluate the accuracy of the test results.
RESULTS
When the plasmid DNA was added to the HTLV-positive samples, the amplified product of IC (400 bp) was detected with the HTLV gene (230 bp). The adequate concentration of plasmid DNA added as an IC was 1 pg.
CONCLUSION
The construction of plasmid DNA as a competitive IC is an efficient method to evaluate accuracy of the test results. However, the production process for the competitive IC must be further developed. Therefore, it is necessary to compare with the performance of a non-competitive IC.

Keyword

HTLV; Laboratory-developed PCR; Internal control

MeSH Terms

Blotting, Western
DNA
Humans
Methods
Nucleic Acid Amplification Techniques
Plasmids
Polymerase Chain Reaction*
T-Lymphocytes
Tissue Donors
DNA