Lin Xin, Flint James A, Azaro Marco, Coradetti Thomas, Kopacka Wesley M, Streck Deanna L, Wang Zhuying, Dermody James, Mandecki Wlodek
PharmaSeq, Inc., Monmouth Junction, NJ 08852, USA.
Clin Chem. 2007 Jul;53(7):1372-6. doi: 10.1373/clinchem.2006.081810. Epub 2007 May 17.
We developed and evaluated a genotyping assay for detection of 50 cystic fibrosis (CF) mutations. The assay is based on small (500 microm) electronic chips, radio frequency (RF) microtransponders (MTPs). The chips are analyzed on a unique fluorescence and RF readout instrument.
We divided the CF assay into 4 panels: core, Hispanic, African-American, and Caucasian. We amplified 18 CF transmembrane regulator (CFTR) DNA fragments covering 50 mutations by use of multiplex PCR using 18 CFTR gene-specific primer pairs. PCR was followed by multiplex allele-specific primer extension (ASPE) reactions and hybridization to capture probes synthesized on MTPs. We used 100 ASPE primers and 100 capture probes. We performed fluorescence measurements of hybridized MTP kits and assay analysis using a custom automated bench-top flow instrument.
We validated the system by performing the assay on 23 commercial DNA samples in an internal study and 32 DNA samples in an external study. For internal and external studies, correct calls were 98.8% and 95.7%, false-positive calls 1.1% and 3.9%, and false-negative calls 0.12% and 0.36%, respectively.
The MTP-based multiplex assay and analysis platform can be used for CF genotyping.
我们开发并评估了一种用于检测50种囊性纤维化（CF）突变的基因分型检测方法。该检测方法基于小型（500微米）电子芯片、射频（RF）微转发器（MTP）。这些芯片在一台独特的荧光和射频读数仪器上进行分析。
我们将CF检测分为4个面板：核心面板、西班牙裔面板、非裔美国人面板和白种人面板。我们使用18对CFTR基因特异性引物对，通过多重PCR扩增18个覆盖50种突变的CF跨膜调节因子（CFTR）DNA片段。PCR之后进行多重等位基因特异性引物延伸（ASPE）反应，并与在MTP上合成的捕获探针杂交。我们使用了100条ASPE引物和100个捕获探针。我们对杂交的MTP试剂盒进行荧光测量，并使用定制的自动化台式流动仪器进行检测分析。
我们通过在一项内部研究中对23个商业DNA样本以及在一项外部研究中对32个DNA样本进行检测，验证了该系统。对于内部和外部研究，正确分型率分别为98.8%和95.7%，假阳性率分别为1.1%和3.9%，假阴性率分别为0.12%和0.36%。
基于MTP的多重检测和分析平台可用于CF基因分型。
