Kit for detecting 33 respiratory pathogens through MALDI-TOF mass spectrum platform

A respiratory and kit technology, which is applied in the field of kits for the detection of 33 respiratory pathogens on the MALDI-TOF mass spectrometry platform, can solve the problems that respiratory pathogens are not easy to quickly realize and cannot meet clinical needs, achieving both flexibility and scalability , low labor cost and detection cost, and simple operation

Inactive Publication Date: 2021-06-04
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current clinical screening for other respiratory pathogens is not easy to achieve quickly
Regardless of whether it is an infection of...

Method used

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  • Kit for detecting 33 respiratory pathogens through MALDI-TOF mass spectrum platform
  • Kit for detecting 33 respiratory pathogens through MALDI-TOF mass spectrum platform
  • Kit for detecting 33 respiratory pathogens through MALDI-TOF mass spectrum platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0250] Example 1 A nucleic acid detection kit for detecting 33 kinds of respiratory pathogens

[0251] 1. Composition

[0252] Multiplex PCR amplification primers and single base extension primers, negative quality control (RNasP(+), 33 pathogens (-)) and positive quality control (RNasP(+), FluA(+), SP(+), other pathogens (-)), one-step multiplex PCR amplification reagents (including: multiplex PCR buffer, multiplex PCR enzyme mixture), de-dNTPs reaction reagents (including: phosphatase buffer, phosphodigestive enzyme) and single base extension reaction reagents (including: extension termination mixture, reaction catalytic enzyme, extension buffer), chip reagents (including: desalting resin, mass spectrometry chip).

[0253] Wherein, multiple PCR amplification primers include:

[0254] (1) Primers for specifically amplifying a certain sequence of the novel coronavirus ORF1ab gene,

[0255] F1: 5'-ACGTTGGGATGCCCTGTGGACTTAAGTTTTAC-3' (SEQ ID NO: 1),

[0256] R1: 5'-ACGTTGGAT...

Embodiment 2

[0465] The detection of embodiment 2 samples

[0466] 1. Experimental method

[0467] According to the method of Example 1, the kit of Example 1 was used to detect 3, 10 samples from different sources and known whether they were infected with the above-mentioned 33 kinds of respiratory pathogens, and the sample numbers were 1 to 10.

[0468] 2. Experimental results

[0469] The results are shown in Table 13. The kit in Example 1 detected 10 samples of known pathogens and the results were consistent with known conditions, with a coincidence rate of 100%. figure 1 Be negative sample representative mass spectrogram in embodiment 2; Figure 2 to Figure 6 It is the positive representative mass spectrum of samples of respiratory syncytial virus B, influenza A virus, mycoplasma pneumoniae, parainfluenza virus I and human coronavirus HCoV-229E in Example 2.

[0470] Table 13 Kit of the present invention detects 10 routine known pathogen sample results

[0471]

[0472]

Embodiment 3

[0473] Sensitivity and accuracy small sample evaluation of embodiment 3 kit

[0474] 1. Experimental method

[0475] 1. Sample selection

[0476] Source and type of samples: 30 cases of nucleic acid samples of clinically confirmed pathogens were selected, including pathogens including novel coronavirus, respiratory syncytial virus (RSVA), and Streptococcus pneumoniae (SP), and 6 cases of plasmid samples, including pathogen detection sites Points are respectively SARS-CoV-2-1ab, LP, SP, HPIV1, HCoV-OC43 and SARS-CoV-2-E, plasmid samples were diluted with 5 gradients (10 2 copies / mL, 10 3 copies / mL, 10 4 copies / mL, 10 5 copies / mL, 10 6 copies / mL) for later use.

[0477] 2. Sample detection

[0478] The above samples were detected using the kit of Example 1 according to the method of Example 1.

[0479] 2. Experimental results

[0480] Table 14:

[0481]

[0482]

[0483] Table 15:

[0484]

[0485] The results are shown in Table 14 and Table 15. The results s...

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Abstract

The invention discloses a kit for simultaneously detecting 33 respiratory tract pathogens through a single reaction hole of an MALDI-TOF mass spectrum platform. The kit comprises multiple PCR amplification primers and single-basic-group extension primers. Other markers such as fluorescein and biotin do not need to be added into the designed primer groups, and the good sensitivity, stability and accuracy are achieved in detection of pathogen nucleic acid sequences; the MALDI-TOF mass spectrum platform has extremely high accuracy and extremely high speed, both sample loading detection and result analysis can be automated, a reaction system only needs common oligonucleotides and does not need fluorescence labeling, the labor cost and the detection cost are low, and the platform is suitable for large-scale population molecular screening and conventional gene diagnosis; and according to the kit, the 33 respiratory pathogens are detected in the same reaction system, the flexibility and expandability are both realized, the operation is simple, the flux is high, the cost is low, and the methodological research trend and the practical demand of the core problem of rapid detection of pathogen nucleic acid can be effectively met.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a kit for detecting 33 kinds of respiratory pathogens on a MALDI-TOF mass spectrometry platform. Background technique [0002] The new coronavirus (SARS-CoV-2) is a new type of highly infectious coronavirus, which belongs to the C lineage of the β-type coronavirus, and the gene size is about 29.8kb. Sequence analysis of the S protein of SARS-CoV-2 shows that although there are differences with the S protein of SARS-CoV, the homology is very high. Compared with the S protein of SARS-CoV, 27 amino acid substitutions occurred, among which there were 6 substitutions at positions 357-528 on the receptor-binding domain (RBD) of S1, and the supporting subunits at positions 569-655. District has 6 replacements. However, SARS-CoV-2 is different from other β-coronaviruses in that the RBD structure of its S protein is closer to the central part of the trimer, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12N15/11C12R1/93C12R1/46C12R1/35C12R1/445C12R1/385C12R1/22C12R1/01
CPCC12Q1/701C12Q1/705C12Q1/689C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2533/101C12Q2565/627C12Q2545/101
Inventor 朱安娜许旭平李明李晓敏刘启祥李婕杨学习
Owner GUANGZHOU DARUI BIOTECH
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