A SYBR green-based real-time PCR method for detection of haemopoietic chimerism in allogeneic haemopoietic stem cell transplant recipients.
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.


CONCLUSIONS
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.
Bai
Lijun
L
Department of Haematology, St Vincent's Hospital, Sydney, NSW, Australia.
Deng
Yi-Mo
YM
Dodds
Anthony J
AJ
Milliken
Sam
S
Moore
John
J
Ma
David D F
DD
D000328:Adult; D005260:Female; D005456:Fluorescent Dyes; D006410:Hematopoiesis / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D019520:Living Donors; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D020080:Tandem Repeat Sequences / Q000235:genetics*; D018183:Transplantation Chimera / Q000235:genetics*; D014184:Transplantation, Homologous
D005456:Fluorescent Dyes