Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

Abstract

The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel “hinge-primer” approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity.

Microfluidic System for Detection of Viral RNA in Blood Using a Barcode Fluorescence Reporter and a Photocleavable Capture Probe

“…28 In addition, our amplification-free protocol can be extended for multiplexing detection which is challenging when performing isothermal amplification due to the interactions of the large number of primers in the assay. 10 …”

“…28 In addition, our amplification-free protocol can be extended for multiplexing detection which is challenging when performing isothermal amplification due to the interactions of the large number of primers in the assay. 10 In the future, it should be possible to reengineer and integrate the current system to provide rapid and sensitive detection at POC without using expensive instruments. Since the SPM, fluorometer, and UV lamp are small in size and low cost, they can be integrated in a small instrument enabling realtime detection of viruses in blood.…”

“…9 In addition, multiplex detection is often limited when using isothermal amplification due to the primer–primer interactions. 10 Thus, a rapid, sensitive, low cost, and fully integrated analysis process which can work with raw blood sample is needed to address POC viral testing needs.…”

“…Isothermal amplification, on the other hand, provides a simple way for performing target amplification and shows excellent sensitivity. , However, nucleic acid prepurification is required because organic and inorganic substances can inhibit the target amplification . In addition, multiplex detection is often limited when using isothermal amplification due to the primer–primer interactions . Thus, a rapid, sensitive, low cost, and fully integrated analysis process which can work with raw blood sample is needed to address POC viral testing needs.…”

Prospects For the Use of Loop Isothermal Amplification in the Diagnosis of Particularly Dangerous Infectious Diseases Caused by the Viruses of the Pathogenicity Group I

“…за последние 30 лет разработан ряд альтернативных способов амплификации нуклеиновых кислот в изотермических условиях. наибольшее развитие и популярность приобрели методы, основанные на цепь-вытесняющей активности некоторых днк-полимераз: амплификация с вытеснением цепи (strand displacement amplification, SDA) [1], амплификация с множественным вытеснением (multiple displacement amplification, MDA) [2], полимеразная спиральная реакция (polymerase spiral reaction, PSR) [3], петлевая амплификация (hinge-initiated primerdepend amplification, HIP) [4] и др. однако наибольшее практическое применение получила петлевая изотермическая амплификация (loop-mediated isothermal amplification, LAMP), которой и посвящен данный обзор.…”