PCR detection method of multiple-target nucleic acid in single pipe and kit thereof

A detection kit and multiple detection technology, applied in the field of nucleic acid detection, can solve the problems of high cost, complicated operation, and aggravated mutual interference.

Active Publication Date: 2010-01-13
SUZHOU SYM BIO LIFESCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when traditional and improved real-time PCR (especially real-time fluorescent PCR) detects actual samples, even if only a single target nucleic acid is detected, it must face the interference of multiple nucleotides that may exist in the sample to the detection results; When performing multiple target nucleic acid detection in a single PCR reaction container (i.e. simultaneously detecting a variety of target nucleic acids), since primers and probes for detecting different target nucleic acids are introdu

Method used

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  • PCR detection method of multiple-target nucleic acid in single pipe and kit thereof
  • PCR detection method of multiple-target nucleic acid in single pipe and kit thereof
  • PCR detection method of multiple-target nucleic acid in single pipe and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 double real-time PCR reaction

[0031] 1. DNA target nucleic acid double Real Time (real time) PCR reaction

[0032]Used to detect HBV DNA and DNA internal control nucleic acid (Seq ID No: 16)

[0033] Primer

[0034] The primers for amplifying HBV DNA are HBV-FQ-L1 (Seq ID No: 1) and HBV-FQ-R1 (Seq ID No: 2); the primers for amplifying the DNA internal control nucleic acid are IC309L1 (Seq ID No: 7) and IC309R1 (Seq ID No: 8)

[0035] fluorescent probe

[0036] The nucleotide sequence of the fluorescent probe of HBV is HBV-FQ-P1 (Seq ID No: 11), and its 5' end is marked with FAM, and the 3' end is marked with a corresponding quenching group; The nucleotide sequence is IC-FQ-P1 (Seq ID No: 14), its 5' end is marked with ROX, and its 3' end is marked with a corresponding quencher group.

[0037] PCR reaction conditions

[0038] The total volume of the PCR reaction system is 60 μl, and the concentrations of each component are: Tris.HCl pH8.3 10mM; KCl 5...

Embodiment 2 3

[0070] Example 2 triple real-time PCR reaction

[0071] 1. RNA target nucleic acid triple real-time PCR reaction

[0072] Used to detect HCV RNA, HIV-1 RNA and RNA internal control nucleic acid (the transcribed DNA sequence is the same as Seq ID No: 17)

[0073] Primer

[0074] The primers for amplifying HCV RNA are HCV-F-L1 (Seq ID No: 3) and HCV-F-R1 (Seq ID No: 4); the primers for amplifying HCV RNA are HIV-F-L1 (Seq ID No: 5) and HIV-F-R1 (Seq ID No: 6); the primers for amplifying the RNA internal control nucleic acid are IC-F-L2 (Seq ID No: 9) and IC-F-R2 (Seq ID No: 10) .

[0075] fluorescent probe

[0076] The nucleotide sequence of the fluorescent probe of HCV is HCV-FQ-P1 (Seq ID No: 12), and its 5' end is labeled with FAM, and the 3' end is marked with a corresponding quenching group; the nucleus of the fluorescent probe of HIV The nucleotide sequence is HIV-F-P1 (Seq ID No: 13), its 5' end is marked with ROX, and the 3' end is marked with the corresponding quen...

Embodiment 3 4

[0084] Embodiment 3 quadruple real-time PCR reaction

[0085] 1. DNA / RNA target nucleic acid quadruple real-time PCR reaction

[0086] 1.1 Used to detect HBV DNA, HCV RNA, DNA internal control nucleic acid (Seq ID No: 16) and RNA internal control nucleic acid (the transcribed DNA sequence is the same as Seq ID No: 17)

[0087] 1.1.1 Primers

[0088] The primers for amplifying HBV DNA are HBV-FQ-L1 (Seq ID No: 1) and HBV-FQ-R1 (Seq ID No: 2); the primers for amplifying HCV RNA are HCV-F-L1 (Seq ID No: 3) and HCV-F-R1 (Seq ID No: 4); the primers for amplifying the internal control nucleic acid of DNA are IC309L1 (Seq ID No: 7) and IC309R1 (Seq ID No: 8); the primers for amplifying the internal control nucleic acid of RNA The primers are IC-F-L2 (Seq ID No: 9) and IC-F-R2 (Seq ID No: 10).

[0089] 1.1.2 Fluorescent probes

[0090] The nucleotide sequence of the fluorescent probe of HBV is HBV-FQ-P1 (Seq ID No: 11), its 5' end is labeled with FAM, and the 3' end is labeled with ...

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Abstract

The invention relates to a real-time PCR method of one or more kinds of target nucleic acid in multiple-detection samples in a signal PCR reaction vessel, which comprises the following steps: (1) mixing a sample, DNA polymerase, dNTP, internal contrast nucleic acid, a target nucleic acid primer pair, an internal contrast nucleic acid primer pair, one or more kinds of target nucleic acid probes and internal contrast nucleic acid probes in the signal PCR reaction vessel, wherein the probes are marked with fluorescent groups and quenching groups, and moreover, the fluoroscopic detection wavelengths of the fluorescent groups marked by various probes are different; (2)detecting fluorescent light with different wave lengths in real time to carry out the PCR reaction; (3) calculating a Ct value according to a fluorescent light detecting result to judge whether one or more kinds of target nucleic acid is stored in the sample; in additon, the invention also discloses a detection kit for the method and preparation, application, and the like of the corresponding detection kit. The multiple-detection method does not need special devices and can be widely used in the fields of laboratory investigation, food security, medicine, hygiene, and the like.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, in particular, the invention relates to a PCR method for multiple detection of target nucleic acids in a sample in a single PCR reaction vessel. In addition, the present invention also relates to the reagents involved in the above PCR method, such as internal control, primers, probes, etc., as well as the detection kit used in the above method and the preparation and application of the corresponding detection kit. Background technique [0002] Polymerase chain reaction (PCR) is one of the most commonly used technologies in nucleic acid detection today. Among them, real-time (Real Time) PCR technology uses real-time detectable markers such as fluorescent markers. It is widely used in nucleic acid detection, clinical diagnosis and molecular biology research. functions are becoming more and more important. This type of technology is currently applied in a wide range of fields, and ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64C12Q1/70C12R1/93
Inventor 张文艳叶成果龚华斐杨国翠李振勇黄道培
Owner SUZHOU SYM BIO LIFESCI CO LTD
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