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Preparation method for nucleic acid quality control by chimeric exogenous independent sequence

A sequence and exogenous technology, applied in biochemical equipment and methods, DNA preparation, microbial measurement/inspection, etc., can solve the problems of low identification accuracy and complex identification reaction, and achieve good reactivity

Active Publication Date: 2014-12-17
重庆海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

What the latter two have in common is that none of the prepared quality control samples is infectious, but the main chimera is the nucleic acid sequence of the test object itself (or similar) and 1~2 common restriction sites to identify and detect False positives and true positives in the results, the identification accuracy is low, and the identification reaction is complicated

Method used

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  • Preparation method for nucleic acid quality control by chimeric exogenous independent sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1, see figure 1 Schematic diagram of the preparation of nucleic acid quality control of chimeric exogenous unrelated sequences, when the detection reaction is based on the length of the product, use method I; when method I cannot meet the actual needs, especially the detection reaction is taqman real-time quantitative PCR or similar probes in the middle When detecting the reaction, use method II.

[0024] (1) Selection of irrelevant sequence sources

[0025] The method I: the irrelevant sequence comes from the existing plasmid vector, such as pUC18, pET30 and so on. Select a DNA sequence from an existing plasmid vector, and use biological software to analyze the GC content and secondary conformation of the DNA sequence. The DNA sequence that meets the following conditions is selected as the irrelevant sequence I: a) The irrelevant sequence I is compatible with the microorganisms to be detected. The homology is less than 5%, b) the GC content is between 45% and...

Embodiment 2

[0039] Example 2, identification of quality: Both the DNA quality control and the RNA quality control can measure OD260, OD260 / OD280 ratio by spectrophotometer and agarose electrophoresis to determine their concentration. It is also necessary to use PCR and RT-PCR to identify the validity of the quality control nucleic acid respectively. Good-quality quality control DNA and RNA should have clear electrophoresis bands, OD260 / OD280 between 1.8 and 2.0, and the amplification curve of the detection reaction is normal.

Embodiment 3

[0040] Embodiment 3, the application of quality control

[0041] (1) Quality control PCR / reverse transcription PCR detection reaction

[0042] DNA quality control and RNA quality control respectively quality control PCR / reverse transcription PCR (RT-PCR) detection reaction. Specifically, for any molecular diagnostic kit, the DNA quality control and the RNA quality control can be used as samples to be tested for the detection reaction of the kit, respectively, and the detection reaction can be carried out according to the instructions of the kit. If the quality control sample has a positive test result, it indicates that the kit is well preserved and the test reaction is established; if the quality control sample shows a negative test result, it indicates that the kit reagents are invalid or the test personnel have performed poorly, and the test reaction has failed.

[0043] (2) Differentiate between true positive amplification and false positive amplification

[0044] If the...

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Abstract

The invention discloses a preparation method for nucleic acid quality control by a chimeric exogenous independent sequence, which is characterized in that a microbiologic specific primer sequence is added by a gene clone method or a chemical synthesis method at two sides of the independent sequence, PCR amplification is used, the amplification product is connected with a pGEM-T carrier to obtain the DNA quality control, and then the RNA quality control which is taken as a molecule detection kit can be obtained by using an in vitro transcription. The prepared nucleic acid quality control contains a segment of gene sequence which is independent to the detection object, in the subsequent positive investigation, whether the false amplification is caused by the operation pollution or not can be identified, under the condition of normal negative control, the positive amplification of the nucleic acid quality control sample indicates the effectiveness and the correctness of the detection reaction.

Description

technical field [0001] The invention belongs to the field of detection of microorganisms (viruses, bacteria, mycoplasma, fungi, etc.) by means of molecular biology, and specifically relates to a set of technical methods for preparing and synthesizing chimeric exogenous irrelevant sequence DNA or RNA quality control. Background technique [0002] Nucleic acid-based molecular biology detection methods or detection kits all need to add standard positive quality control samples to test the effectiveness of the detection process. However, the addition of standard positive samples all derived from the detection object often affects the detection reaction. Potential contamination and interpretation interference. At present, the main quality control samples used in molecular diagnostic kits are virus serum with infectious activity, such as the hepatitis C virus standard quality control prepared by Wang Lunan et al.: Dilute the positive plasma to about 300,000 copies with HCV RNA neg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 王昱肖进文李贤良聂福平李应国杨俊
Owner 重庆海关技术中心
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