A long downstream probe-based platform for multiplex target capture

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• 作者：Ping Wang^a ; ^b ; Jianqin Huang^a ; Yingwu Xu^a ; ^{yxu@zafu.edu.cn" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Suppressor ; Single-strand long downstream probe ; Ligase detection reaction ; Stuffer sequence ; Concordance rates
• 刊名：Analytical Biochemistry
• 出版年：2015
• 出版时间：15 December 2015
• 年：2015
• 卷：491
• 期：Complete
• 页码：4-9
• 全文大小：1192 K

文摘

A simple and rapid detection platform was established for multiplex target capture through generating single-strand long downstream probe (ssLDP), which was integrated with the ligase detection reaction (LDR) method for the purpose of multiplicity and high specificity. To increase sensitivity, the ladder-like polymerase chain reaction (PCR) amplicons were generated by using universal primers that complement ligated products. Each of the amplicons contained a stuffer sequence with a defined yet variable length. Thus, the length of the amplicon is an index of the specific suppressor, allowing its identification via electrophoresis. The multiplexed diagnostic platform was optimized using standard plasmids and validated by using potato virus suppressors as a detection model. This technique can detect down to 1.2 × 10³ copies for single or two mixed target plasmids. When compared with microarray results, the electrophoresis showed 98.73–100% concordance rates for the seven suppressors in the 79 field samples. This strategy could be applied to detect a large number of targets in field and clinical surveillance.