Quantitative Multiplex Real-Time Reverse Transcriptase-Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3

Wipaporn Wongfieng; Rungtiwa Nutalai; Amonrat Jumnainsong; Chanvit Leelayuwat

doi:10.3390/diagnostics10080588

Quantitative Multiplex Real-Time Reverse Transcriptase-Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3

Diagnostics (Basel). 2020 Aug 13;10(8):588. doi: 10.3390/diagnostics10080588.

Authors

Wipaporn Wongfieng^{1

2}, Rungtiwa Nutalai^{2

3}, Amonrat Jumnainsong^{2

4}, Chanvit Leelayuwat^{2

4}

Affiliations

¹ Research Administration Division of Khon Kaen University, Khon Kaen 40002, Thailand.
² The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
³ Biomedical Sciences Program, Graduates School of Khon Kaen University, Khon Kaen 40002, Thailand.
⁴ Department of Clinical Immunology and Transfusion Sciences, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.

Abstract

(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection-especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 10⁷ copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R² = 0.995, KIR3DL3; R² = 0.996, but lower sensitivity of 10³ copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications.

Keywords: fluorescent dyes; killer-cell immunoglobulin-like receptors; limit of detection; qRT-PCR.

Abstract

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