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Primer-probe combination and detection kit for detecting new coronavirus and pneumonia pathogenic bacteria

A technology for detection kits and primer probes, applied in the field of primer probe combinations and detection kits for the detection of novel coronavirus and pneumonia pathogenic bacteria, to achieve high sensitivity, good sensitivity and specificity, efficient screening and triage

Active Publication Date: 2021-12-31
上海思路迪医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It aims to solve the problem that the triage of new coronavirus infection and community-acquired pneumonia cannot be carried out through one test in the existing technology

Method used

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  • Primer-probe combination and detection kit for detecting new coronavirus and pneumonia pathogenic bacteria
  • Primer-probe combination and detection kit for detecting new coronavirus and pneumonia pathogenic bacteria
  • Primer-probe combination and detection kit for detecting new coronavirus and pneumonia pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Preparation of detection kits for novel coronavirus and pneumonia pathogens

[0089] Prepare nucleic acid amplification reaction solution according to Table 2 and Table 3 below.

[0090] Table 2: Components and concentrations of nucleic acid amplification reaction solution A

[0091]

[0092] Table 3 Component B and concentration of nucleic acid amplification reaction solution

[0093]

[0094] Prepare the enzyme mixture according to Table 4.

[0095] Table 4

[0096]

[0097] Positive controls were prepared according to Table 5.

[0098] table 5

[0099]

[0100] Prepare negative control and blank control according to Table 6.

[0101] Table 6

[0102] name Component concentration Loading volume (μL) negative control Human Embryonic Kidney Cell 293T Culture Medium 5×10 4 cell / mL

Embodiment 2

[0103] Example 2 The method of using the new coronavirus and pneumonia pathogenic bacteria detection kit

[0104] The method for using the detection kit of the present invention comprises the following steps:

[0105] (1) PCR reaction solution preparation

[0106] Take out nucleic acid amplification reaction solution A, nucleic acid amplification reaction solution B and enzyme mixture from the kit, thaw at room temperature, shake and mix well, according to the number of samples to be amplified N (including positive control, negative control and blank control) 7 and Table 8 respectively prepare the PCR reaction solution A tube and the PCR reaction solution B tube, mix thoroughly, centrifuge for a short time and dispense into PCR reaction tubes (plates) with a volume of 12.5 μL.

[0107] Table 7 Preparation of tube A of PCR reaction solution

[0108] component name Volume (μL) Reaction solution preparation Nucleic Acid Amplification Reaction Solution A 11 ...

Embodiment 3

[0121] Example 3 Sensitivity of the kit of the present invention for detection

[0122] Using the kit of Example 1, detect the national standard product 2019-nCoV nucleic acid detection reagent national reference product (370099-202001), verify the detection sensitivity; the sensitivity reference product S concentration (stock solution) is 3 × 10 5 Copies / mL, jointly determined by digital PCR method. S using RNA / DNase-free deionized water for 1:3 dilution (2 parts water + 1 sample), 1:9, 1:27, 1:81, 1:243, 1:729, 1 :2187, 1:6561, 1:19683, 1:59049, and 1:177147 were marked as S1-S10 respectively, and were detected after nucleic acid extraction according to the kit instructions. According to the test results after serial dilution, the detection sensitivity of this kit to SARS-CoV-2 is 0.137 copies / μL (S6), such as Figure 3-Figure 4 shown, where image 3 Schematic diagram of the detection sensitivity of the novel coronavirus ORF1ab gene; Figure 4 It is a schematic diagram o...

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Abstract

The invention discloses a primer-probe combination and detection kit for detecting new coronavirus and pneumonia pathogenic bacteria. A primer pair for detecting a new coronavirus ORF1ab gene is shown as SEQ ID NO: 1-2, and a probe is shown as SEQ ID NO: 3; a primer pair for detecting a new coronavirus N gene is shown as SEQ ID NO: 4-5, and a probe is shown as SEQ ID NO: 6; and a primer pair for detecting haemophilus influenzae is shown as SEQ ID NO: 7-8, and a probe is shown as SEQ ID NO: 9. A primer pair for detecting streptococcus pneumoniae is shown as SEQ ID NO: 10-11, and a probe is shown as SEQ ID NO: 12; a primer pair for detecting mycoplasma pneumoniae is shown as SEQ ID NO: 13-14, and a probe is shown as SEQ ID NO: 15; and a primer pair for detecting chlamydia pneumoniae is shown as SEQ ID NO: 16-17, and a probe is shown as SEQ ID NO: 18. The primers and the probes provided by the invention have good sensitivity and specificity, and can be efficiently applied to screening and triage of new coronavirus infection and community-acquired pneumonia infection by applying the primers and the probes to the detection kit.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a combination of primers and probes and a detection kit for detecting novel coronavirus and pneumonia pathogenic bacteria. Background technique [0002] The novel coronavirus (COVID-19) has been listed as a Class B infectious disease and managed as a Class A infectious disease. In order to guide local disease control agencies to standardize the epidemiological investigation of new coronavirus pneumonia, grasp the epidemiological information such as case incidence, exposure history, and contact history, and analyze the transmission characteristics and transmission chains of clustered epidemics, do a good job in close contact. Contact tracing and determination to prevent the spread and spread of the novel coronavirus pneumonia epidemic, according to legal requirements, all cases should be reported immediately through the infectious disease information syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/689C12Q1/6848C12Q2600/166C12Q2521/107C12Q2521/531C12Q2531/113C12Q2545/101C12Q2527/127
Inventor 张琳巫益鸣
Owner 上海思路迪医学检验所有限公司
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