A long downstream probe-based platform for multiplex target capture.
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 × 10(3) 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.
Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an 311300, China; National Center for Protein Science Shanghai, Shanghai 201210, China.
Ping
P
Wang
Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an 311300, China.
Jianqin
J
Huang
Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an 311300, China. Electronic address: yxu@zafu.edu.cn.
Yingwu
Y
Xu
D017931:DNA Primers / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis / Q000378:metabolism; D019075:Electrophoresis, Capillary; D010942:Plant Viruses / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins
Concordance rates; Ligase detection reaction; Single-strand long downstream probe; Stuffer sequence; Suppressor