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Mismatched fluorescence tolerance real-time quantitative PCR method for detecting multiple pathogens

A real-time quantification of pathogens, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of primer and template mismatch, increase the difficulty of detection, error detection results, etc., to achieve maximum convenience and detection results Accurate and reliable, easy and fast detection operation

Pending Publication Date: 2019-04-23
南京迈维生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the variety of microorganisms such as viruses and bacteria, which mutate rapidly, there will be a large number of variants in the sample, so mismatches between primers and templates are prone to occur, resulting in errors in detection results (such as false positives, false negatives)
In order to detect multiple types of viruses or different virus subtypes, strains or variants of the same type of virus, separate tests are often required, which increases the difficulty of detection and is time-consuming and laborious

Method used

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  • Mismatched fluorescence tolerance real-time quantitative PCR method for detecting multiple pathogens
  • Mismatched fluorescence tolerance real-time quantitative PCR method for detecting multiple pathogens
  • Mismatched fluorescence tolerance real-time quantitative PCR method for detecting multiple pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Detection of RSV wild-type templates and mutant templates based on common primer pairs, a small amount of high-fidelity DNA polymerase and non-high-fidelity DNA polymerase.

[0049] This embodiment has obtained RSV virus wild-type template (RSV-W, SEQ ID NO.1), made 2 templates (RSV-M-T, SEQ ID NO.2) and (RSV-M- AC, SEQ ID NO.3), the mutation sites are the template positions corresponding to the 3' end of the upstream primer, and one common upstream primer (RSV-F, SEQ ID NO.5) and one common downstream primer (RSV-F, SEQ ID NO.5) were designed ( RSV-R, SEQ ID NO.6) and probe (RSV-P, SEQ ID NO.7), the 5' end of the probe is labeled with VIC fluorescent group, and the 3' end is labeled with MGB quenching group. Common upstream primers, downstream primers, and probes are perfectly complementary to wild-type templates.

[0050] Use non-high-fidelity DNA polymerase (Taq) and high-fidelity DNA polymerase (Q5) to apply to the amplification reaction. The reaction sy...

Embodiment 2

[0058] Example 2. Based on two common primers and one probe, a large amount of non-high-fidelity DNA polymerase and a small amount of high-fidelity DNA polymerase are used to detect Boca virus.

[0059] In this example, a recombinant plasmid inserted into a partial fragment of Boca virus was obtained as a wild-type template (BOV-W, SEQID NO.4), and two upstream primers (bov-F-M1, bov-F-M1, bov -F-M2, SEQ ID NO.8, SEQ ID NO.9), 1 common upstream primer (bov-F, SEQ ID NO.10) and 1 common downstream primer (bov-R, SEQ ID NO.11), The first T at the 3' end of one of the upstream primers was mutated to A (bov-F-M1), and the first and second T at the 3' end of the other upstream primer were both mutated to A (bov-F-M2). The 5' end of the needle is labeled with the FAM fluorophore, and the 3' end is labeled with the BHQ1 quencher. The common upstream primer, downstream primer and probe (bov-P, SEQ ID NO.12) are fully complementary to the wild-type template (BOV-W).

[0060] Use non-...

Embodiment 3

[0068] Example 3. Using the new method and the traditional Taqman method to simultaneously detect 10 RSV positive clinical samples.

[0069] The present embodiment has obtained 20 parts to determine the nucleic acid of the clinical sample of positive RSV, with the designed RSV upstream primer (RSV-F, SEQ ID NO.5), downstream primer (RSV-R, SEQ ID NO.6) and The probe (RSV-P, SEQ ID NO.7) was detected by qRT-PCR on 10 samples using the new method and the traditional Taqman method.

[0070] The new method uses non-high-fidelity DNA polymerase (Taq) and high-fidelity DNA polymerase (Q5) for amplification reactions. The reaction system and procedures are as follows:

[0071]

[0072] The reaction conditions are as follows:

[0073]

[0074] The traditional Taqman method does not add Q5, and the remaining components and reaction conditions of the system are the same as the new method.

[0075] According to the above reaction system and reaction conditions in ROCHE The reac...

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Abstract

The invention provides a mismatched fluorescence tolerance real-time quantitative PCR method for detecting multiple pathogens and an application in virus (particularly RNA virus with high variability)detection. The invention relates to a DNA polymerase (non-high fidelity DNA polymerase) without a 3'->5'exonuclease correction function and a high fidelity DNA polymerase with the 3'->5'exonuclease correction function, a primer is driven to extend under the action of the non-high fidelity DNA polymerase when no mismatch is caused to form a DNA product. When the 3'end of the primer is mismatched with the template, the high-fidelity DNA polymerase excises the mismatched base at the 3'end of the primer, and a common DNA polymerase is responsible for catalyzing DNA synthesis; the method has the advantages of good specificity, high sensitivity, good repeatability, simple operation, convenience and rapidness, and can be widely applied to the related fields of virus detection and the like.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to a mismatch tolerance fluorescent real-time quantitative PCR method for detecting various pathogens. Background technique [0002] Infectious disease virus detection is the key to the prevention and control of emerging infectious diseases, and rapid and accurate detection is especially critical for prevention, treatment and control. [0003] At present, the main methods for laboratory detection of viruses include virus isolation, serum antibody detection and nucleic acid detection. The virus isolation method requires specific experimental conditions and personnel, and the experimental period is long, and it is limited to special purposes and uses. It is currently a widely used rapid detection standard method; the serum antibody detection operation is relatively simple, but the sensitivity is low and there are many interference factors. It needs to be combined with ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/6851C12Q1/701C12Q2563/107C12Q2561/113C12Q2531/113C12Q2521/107
Inventor 乔红兵
Owner 南京迈维生物科技有限公司
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