Frenoy N, Chabli A, Sol O, Goldschmit E, Lemonnier M P, Misset J L, Debuire B
Service de Biochimie, Hôpital Paul Brousse, Villejuif, France.
Leukemia. 1994 Aug;8(8):1411-4.
Nested PCR (NPCR), a two-step procedure in which the products of a first PCR using 'outer' primers are reamplified using 'inner primers', has been successfully used to test for the chronic myeloid leukemia (CML)-specific bcr-abl transcripts. A major drawback of the conventional nesting strategy is linked to the opening of the reaction tube between the two successive PCR reactions, giving a risk of contaminating the second mix with amplicons. In this paper, the application of a new protocol for NPCR without reopening the reaction tube between the two steps of the procedure is described for the research of residual leukemic cells in the peripheral blood of 14 CML patients treated by bone marrow transplantation (BMT) or interferon (IFN). This assay which is both highly specific and sensitive, offers several advantages over the use of conventional NPCR: it is more sensitive, faster and decreases the risk of false-positive results. In addition, chemiluminescent detection of amplified DNA after transfer onto a nylon membrane, although comparable with radioactive hybridization in terms of sensitivity and speed, is more advantageous in safety and convenience. In conclusion, this assay could be adapted to a number of clinical diagnostic uses.
巢式聚合酶链反应（Nested PCR，NPCR）是一种两步法程序，其中使用“外部”引物进行的第一次PCR产物再用“内部引物”进行扩增，已成功用于检测慢性粒细胞白血病（CML）特异性的bcr-abl转录本。传统巢式策略的一个主要缺点与在两个连续的PCR反应之间打开反应管有关，这会有使第二个反应混合物被扩增子污染的风险。本文描述了一种新的NPCR方案的应用，该方案在程序的两个步骤之间无需重新打开反应管，用于研究14例接受骨髓移植（BMT）或干扰素（IFN）治疗的CML患者外周血中的残留白血病细胞。该检测方法具有高度特异性和敏感性，与使用传统NPCR相比具有几个优点：它更敏感、更快且降低了假阳性结果的风险。此外，将扩增的DNA转移到尼龙膜上后进行化学发光检测，尽管在灵敏度和速度方面与放射性杂交相当，但在安全性和便利性方面更具优势。总之，该检测方法可适用于多种临床诊断用途。
