【摘要】 目的探讨最适退火温度和核酸扩增(PCR)周期,利用筑巢式PCR检测慢性粒细胞白血病(CML)的bcr-abl融合基因类型,并进行PCR荧光定量(RQ-PCR),研究其与疾病的关系。方法通过改变PCR条件,将退火温度由55℃增加到60℃,反应周期由原来的30周期增加到45周期,检测14例22份标本。同时用荧光定量试剂检测标本。结果退火温度为58℃,45周期可测出103copies/μl定量标准品的PCR产物。22份标本bcr-abl融合基因巢式PCR结果均阳性。其中b2a2型9份,b3a2型13份。定量检测18例阳性,量值在102~106copies/μg,两例急变患者急性期和慢性期有明显差异。结论筑巢式PCR是一种敏感而准确的检测方法,对bcr-abl进行定量有助于反映白血病细胞负荷、评价疗效及判断疾病预后。更多还原

【Abstract】 Objective To increase the sensitivity of residual leukemic cells detectin in chronic myelocytic leukemia（CML） patients with RT-PCR,the optional annealing temperature and PCR cycles were studied to confirm bcr-abl fused gene types,and bcr-abl mRNA transcripts were monitored by RQ-PCR to study the relation with CML at different phases. Methods Through changing the PCR conditions, the annealing temperature was measured from 55℃ to 60℃, and the number of reaction cycles was increased from 30 to 45.All 22 samples were examined, and bcr-abl mRNA transcripts were quantified by RQ-PCR kit. Results Bcr-abl fused gene types were found in 22 samples,of all 9 cases were b₂a₂ type, 13 cases were b₃a₂.When the annealing temperature was set for 58℃ and the number of reation cycles was set for 45,10³ copies/ul standard samples was detected.18 samples were positive tested by RQ-PCR kit,and the value was between 10² to 10⁶ copies/g.There were significant differce between the results of chronic phase samples and those of accelerated phase. Conclusions The RT-PCR is a reliable,sensitive and reproducible method of monitoring CML patients.The real-time RT-PCR is useful in evaluating leukemic burden,assessing response to treatment and predicating the prognosis of the disease.更多还原