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A SYBR green‐based real‐time PCR method for detection of haemopoietic chimerism in allogeneic haemopoietic stem cell transplant recipients
Author(s) -
Bai Lijun,
Deng YiMo,
Dodds Anthony J.,
Milliken Sam,
Moore John,
Ma David D. F.
Publication year - 2006
Publication title -
european journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.904
H-Index - 84
eISSN - 1600-0609
pISSN - 0902-4441
DOI - 10.1111/j.1600-0609.2006.00729.x
Subject(s) - transplantation chimera , taqman , biology , real time polymerase chain reaction , polymerase chain reaction , microbiology and biotechnology , microsatellite , transplantation , serial dilution , sybr green i , melting curve analysis , stem cell , genetics , gene , haematopoiesis , medicine , hematopoietic cell , pathology , allele , alternative medicine , surgery
Objective: To develop a reliable and rapid real‐time quantitative polymerase chain reaction (Q‐PCR) method using SYBR green to quantify chimerism in allogeneic haemopoietic stem cell transplant recipients. Methods: Twelve specific nucleotide polymorphisms (NPs) were selected to identify informative markers for detecting chimerism in transplant donor/recipient pairs. One informative marker was then used in SYBR green Q‐PCR to detect chimerism post‐transplantation in each patient. The percentage of donor cells was calculated using a standard curve, constructed using artificially mixed donor/recipient chimeric DNA in 12 serial dilutions (0.01–100%). Results: DNA from 37 donor/recipient pairs was screened for informative markers and 18 post‐transplant samples were monitored for chimerism with SYBR green Q‐PCR method. The Q‐PCR was able to discriminate between recipient and donor genetic profiles in all 18 samples, and quantify the chimerism. These results were confirmed by at least one independent method, such as TaqMan Q‐PCR, microsatellite and fluorescent in situ hybridisation (FISH) methods. The detection limit of this method was 0.1%, which was more sensitive than the two currently used microsatellite and FISH methods. Conclusion: The new single platform SYBR green Q‐PCR method is capable of detecting all haemopoietic chimerism with high accuracy; hence, it may be used to replace the current detection methods.