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Donor Chimerism Study by Single Nucleotide Polymorphism using SYBR green based Real Time PCR.

Ayesha Nayyar1, Suhaib Ahmed2

  • 1Dr. Ayesha Nayyar, M.Phil. Department of Pathology, Islamic International Medical College, Riphah International University, Islamabad, Pakistan.

Pakistan Journal of Medical Sciences
|December 16, 2021
PubMed
Summary

This study optimized a real-time PCR method using Single Nucleotide Polymorphism (SNP) and SYBR Green to detect donor chimerism after hematopoietic stem cell transplantation (HSCT). The sensitive assay accurately monitored chimerism in post-transplant patients.

Keywords:
DCInformative allelePCRSNPSTRSYBR Green

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Area of Science:

  • Molecular Biology
  • Immunogenetics
  • Transplantation Science

Background:

  • Hematopoietic stem cell transplantation (HSCT) is a critical treatment for various hematological disorders.
  • Monitoring donor chimerism post-HSCT is essential for assessing graft success and patient outcomes.
  • Existing methods for chimerism analysis require optimization for sensitivity and efficiency.

Purpose of the Study:

  • To optimize and evaluate a Single Nucleotide Polymorphism (SNP) real-time PCR assay utilizing SYBR Green dye.
  • To assess the utility of this optimized method for detecting donor chimerism after allogeneic HSCT.
  • To compare the performance of SNP real-time PCR with Short Tandem Repeat (STR) PCR for chimerism analysis.

Main Methods:

  • A descriptive study involving 20 patients who underwent allogeneic HSCT from HLA-matched sibling donors.
  • Chimerism status was determined using both SNP real-time PCR with SYBR Green and STR PCR.
  • The study was conducted at the Genetic Resource Centre (GRC) Lab Rawalpindi from October 2017 to December 2019.

Main Results:

  • The SNP SYBR Green real-time PCR successfully detected significant donor chimerism in all 20 post-HSCT patients.
  • The assay demonstrated high precision, with mean donor chimerism values of 94.1% (SD 3.96) by SNP and 95.1% (SD 1.41) by STR PCR.
  • The method exhibited excellent sensitivity, with a detection limit of less than 1%.

Conclusions:

  • SNP analysis employing SYBR Green real-time PCR is a viable and sensitive method for evaluating chimerism status post-HSCT.
  • This sensitive assay can be effectively utilized for monitoring donor chimerism in patients with hematological disorders following transplantation.
  • The optimized real-time PCR method offers a valuable tool for routine clinical assessment of HSCT recipients.