Internal reference detection system and kit for isothermal nucleic acid amplification reaction

Abstract

The invention designs an internal reference detection system, and concretely relates to an internal reference detection system for the isothermal nucleic acid amplification reaction, a kit containing the internal reference detection system and an application of the system. The internal reference detection system is used for detecting the false negative reaction in the isothermal the nucleic acid amplification reaction, and comprises template DNA with a hairpin structure, a specific primer 1 and a specific primer 2, wherein the specific primer 1 is complementary with the sequence of a ring in the hairpin structure, and the specific primer 2 is complementary with the 3'-terminal sequence at the stem in the hairpin structure. The invention also relates to the kit of the internal reference detection system for the isothermal nucleic acid amplification reaction, and the kit comprises a strand displacement-function DNA polymerase-mediated isothermal nucleic acid amplification system and an internal reference detection system. The internal reference detection system of the invention has the advantages of exquisite design, sensitive reaction and suitableness for the large-scale popularization and use.

Description

technical field [0001] The invention designs an internal reference detection system, specifically, relates to an internal reference detection system for nucleic acid constant temperature amplification reaction, a kit containing the internal reference detection system, and applications thereof. Background technique [0002] In recent years, the trend of global epidemics shows that various difficult-to-culture and non-culturable pathogenic microorganisms are becoming the main source of infectious diseases. Conventional pathogen detection methods (such as culture identification) are not suitable for the current clinical detection requirements due to their own shortcomings. However, with the development of molecular biology technology, especially nucleic acid amplification technology, the ability to detect and identify pathogenic bacteria has been greatly improved. [0003] Nucleic acid amplification technology refers to the specific amplification of a large number of sequence ...

AI Technical Summary

Problems solved by technology

Differences in instrument design principles and frequent quality problems (such as inaccurate temperature control, etc.) have brought clinical problems such as non-specific amplification (false positive) and decreased amplification efficiency (false negative)

[0005] 2. Centrifuge problem

The quality or use of the centrifuge is incorrect, for example, the length of the effective radius leads to the difference in the centrifugation effect, which makes the DNA separation of the sample unable to be effectively separated, and eventually leads to false negatives; due to the high-speed rotation of the high-speed centrifuge and air friction will produce a large amount of Heat, resulting in false negatives caused by changes in the amplification reaction system, etc.

[0006] 3. Nucleic acid extraction process: The reagents and operations used in the extraction process will cause the sample DNA not to be extracted, or inhale some inhibitors during the sample loading process, which will make the amplification impossible and false negative results will appear.

[0007] 4. Inhomogeneity of reagent transportation and storage environment can also lead to false negative results

Most of these NAIA systems are nucleic acid amplification mediated by DNA polymerase with strand displacement function, for example, LAMP, HAD, and CPA are mediated by Bst DNA polymerase; RCA and SDA are mediated by Klenow large fragment DNA polymerase, However, there is no corresponding reaction internal reference detection system in these NAIA technologies.

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