ANALYSIS OF MULTIPLEX LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (MLAMP) PRODUCTS BASED ON NICKING MEDIATED SEQUENCE-TAGGED PYROSEQUENCING
The loop-mediated isothermal amplification(LAMP) is widely used in many fields with high-sensitivity and specificity. It is an obvious limit of LAMP that the method is hard to be used in the amplification of multiple targets. In this paper, we proposed a method enabling multiplex loop-mediated isothermal amplification (mLAMP) based on nicking mediated sequence-tagged Pyrosequencing. Recognition sequence of NEases was introduced by FIP primer. LAMP products were treated to remove excess amounts of primers, nucleotides, and pyrophosphate (PPi) prior to sequencing. After the nicking reaction, pyrosequencing starts at the nicked 3 end, and extension reaction occurs when the added dNTP was complementary to the non-nicked strand. Although the activity of strand displacement by Klenow was limited, ～10bases were accurately sequenced; this length was long enough for analysis of the sequencetag which represent different template. Thus, the diagnosis of multiple pathogens in blood such as HBV, HCV, HIV and TP were successfully executed. The results indicate that multiplex loop-mediated isothermal amplification based on nicking sequencetagged Pyrosequencing is a simple, rapid and reliable way in multiple targets analysis.
Liang Chao Chu Yanan Cheng Sijia Zhou Guohua
Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China College of Life Scien Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China
国际会议
重庆
英文
239-243
2011-10-28（万方平台首次上网日期，不代表论文的发表时间）