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Mycobacterium tuberculosis sRNA fluorescent quantitative PCR standard substance for identifying false positive reaction and application thereof

A technique for the quantification of mycobacterium tuberculosis and fluorescence, applied in the field of microbiology

Active Publication Date: 2021-04-20
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a Mycobacterium tuberculosis sRNA fluorescent quantitative PCR standard substance for identifying false positive reactions, and to establish a real-time, fast and accurate nucleic acid quantification method for distinguishing PCR standard substance signals and specific target signals to be detected The PCR detection method overcomes the false positive detection results caused by cross-contamination between standard components and samples to be tested in the existing nucleic acid quantitative PCR detection technology, and improves the specificity and accuracy of nucleic acid quantitative PCR detection

Method used

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  • Mycobacterium tuberculosis sRNA fluorescent quantitative PCR standard substance for identifying false positive reaction and application thereof
  • Mycobacterium tuberculosis sRNA fluorescent quantitative PCR standard substance for identifying false positive reaction and application thereof
  • Mycobacterium tuberculosis sRNA fluorescent quantitative PCR standard substance for identifying false positive reaction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 sRNA standard product sequence and primer design

[0033] Select a wild sequence in the gene sequence of Mycobacterium tuberculosis small RNA MTS0997, and delete some bases, so that the theoretical TM value of the mutated nucleotide sequence is 4-6°C different from that of the wild sequence. Compared with the original sequence, The sRNA standard sequence with a mutation site in the middle of the amplified sequence was named #0997(-21), and was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The full length of the standard is 92nt, and the PCR amplification length is 86nt.

[0034] Table 1 sRNA standard sequence

[0035]

[0036] 2.PCR primer sequence

[0037]The primers used to amplify the #0997(-21) sequence and the MTS0997 sequence in bacteria are the same set, and the primer sequences are shown in Table 2.

[0038] Table 2 Primer sequences for amplifying RNA

[0039]

Embodiment 2

[0040] Embodiment 2 establishes sRNA standard substance real-time quantitative PCR detection system and draws standard curve

[0041] 1. RNA reverse transcription

[0042] (1) Take one tube (0.6 nmol) of sRNA standard #0997(-21) (SEQ ID No.1) dry powder and dissolve it in 100 μl DEPC-treated water. 1mol RNA is 6.02×10 23 copy number (copies), 0.6nmol RNA dry powder standard is 3.6×10 14 Copies, 100μl DEPC-treated water dissolved to 3.6×10 12 copies / μl.

[0043] (2) sRNA standard substance is carried out 10 fold dilutions (10 12 -10 0 copies / μl, 10 μl of RNA standard and 90 μl of DEPC-treated water).

[0044] (3) Take 1 μl sRNA standard of each concentration for reverse transcription.

[0045] Reverse transcription system:

[0046]

[0047]

[0048] The reverse transcription program was: 10 minutes at 25°C, 15 minutes at 42°C, and 5 minutes at 85°C.

[0049] 2. Take 2 μl of cDNA for each dilution factor for qPCR experiment

[0050] (1) qPCR system (including pri...

Embodiment 3

[0059] Stability and repeatability test of embodiment 3 sRNA standard substance

[0060] The standard curve making process of the sRNA standard in Example 2 was repeated three times. The CT values ​​of the three results are shown in Table 3. The experimental results showed that the CVs obtained in three repeated experiments were all less than 1.5%, indicating that the sRNA standard had high stability and repeatability.

[0061] Table 3 Stability and repeatability of sRNA standards

[0062]

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Abstract

The invention discloses a mycobacterium tuberculosis sRNA fluorescent quantitative PCR standard substance for identifying false positive reaction and application thereof. The difference between the theoretical TM value of the mycobacterium tuberculosis sRNA fluorescent quantitative PCR standard substance and the theoretical TM value of a wild sequence is 4-6 DEG C, the pollution caused by the standard substance can be rapidly and accurately monitored in real time in quantitative PCR detection, and it is avoided that cross contamination between a standard substance assembly of a traditional kit and a sample influences accurate quantification of the sample. According to the invention, the standard substance pollution identification is simple and convenient, extra instruments and operation steps are not needed, and any possible standard substance amplification pollution can be rapidly judged in real time only by identifying a quantitative PCR melting curve, so that the manpower and material resources are saved, and the economical efficiency is high.

Description

technical field [0001] The invention relates to the design, preparation and real-time quantitative PCR detection of a Mycobacterium tuberculosis sRNA standard product, which can determine the false positive reaction caused by standard product pollution in the fluorescent quantitative PCR detection of Mycobacterium tuberculosis sRNA. The present invention belongs to the field of microbiology. Background technique [0002] The small RNA (small RNA, sRNA) of Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is a type of RNA with high expression in the bacteria, secondary structure, and stability; in infected tissues, the sRNA of MTB High expression. Studies have shown that in the lung tissue of mice infected with Mycobacterium tuberculosis, the expression levels of MTS0997, MTS1338 and MTS2823, the three Mycobacterium tuberculosis sRNAs, are the highest ([1] DV I, OIu T, NN L, et al. Expression of Mycobacterium tuberculosis small RNAs in mice models of tuberculosis...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11
Inventor 付英梅张凤民李婷孟庆泰宋武琦韩雪
Owner HARBIN MEDICAL UNIVERSITY
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