A multiplex PCR primer and probe combination for detecting pathogens and its application

A technology of pathogens and probes, which is applied to the combination of multiple PCR primers and probes for the detection of 18 pathogens and its application field, which can solve the problems of difficult identification and determination of virus types, harsh virus culture conditions, and inability to cultivate viruses, and achieve convenient and rapid Effectiveness of screening, avoiding false positives, avoiding false negatives

Active Publication Date: 2022-07-01
潮州凯普生物化学有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to identify and determine the type of virus infected through clinical symptoms and routine laboratory tests, and the culture conditions of the virus are relatively harsh, resulting in a low positive culture rate, and even some viruses cannot be cultured under the current conditions. and clinicians a great deal of trouble

Method used

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  • A multiplex PCR primer and probe combination for detecting pathogens and its application
  • A multiplex PCR primer and probe combination for detecting pathogens and its application
  • A multiplex PCR primer and probe combination for detecting pathogens and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Multiplex PCR primer and probe combination

[0083] The present invention uses influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, human parainfluenza virus (type I, type II, type III), enterovirus, dengue virus, eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, forest encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, Nipah virus and Hendra virus representative strains, the nucleotides of these 18 pathogens The nucleotide sequences of the representative strains of the above infectious pathogens were downloaded from the NCBI database. Through multiple sequence comparison and analysis, multiple PCR primers and probes that can be used to detect the above pathogens were designed, and an internal standard was also designed. Primers, internal standard probes, and chromogenic system control probes are used to monitor sample collection and extracti...

Embodiment 2

[0090] Example 2 Gene chip and detection kit

[0091] Based on the multiplex PCR primers and probes described in Example 1, the present invention develops gene chips and detection kits for detecting the above 18 pathogens. The gene chip includes an immobilized carrier and a pathogen-specific probe immobilized on the immobilized carrier, namely the probe sequences shown in SEQ ID No. 29 to 46, and also contains the internal standard probe sequence shown in SEQ ID No. 49 and The chromogenic system shown in SEQ ID No. 50 controls the sequence of the probe. The probe is synthesized and modified by a primer synthesis manufacturer (Shanghai Jierui Bioengineering Co., Ltd.), and the 5' and 3' ends of the probe are amino groups. Chemical treatment (amino group Amino), the color system control probe is labeled with biotin dots.

[0092] 1. Preparation of gene chip

[0093] (1) Arrangement of probes

[0094] In this example, the fixed carrier used in the preparation of the gene chip ...

Embodiment 3

[0104] Example 3 Detection method of infectious disease pathogens

[0105] The present invention takes the kit described in Example 2 as an example to briefly describe the method for detecting the 18 pathogens using the kit for the purpose of non-disease diagnosis.

[0106] details as follows:

[0107] (1) release the nucleic acid of the sample to be tested;

[0108] (2) RT-PCR amplification;

[0109] Use primer pairs 1 to 15 described in the kit to perform RT-PCR amplification on the samples to be tested. The PCR reaction volume is 45 μL, the DNA sample volume is 5 μL, and the total reaction volume is 50 μL. The reaction system of PCR amplification is shown in Table 3:

[0110] Table 3 Multiplex PCR reaction system

[0111]

[0112] Note: Q-solution is an auxiliary reagent for gene amplification. The 5' ends of the primers are labeled with fluorescein. The enzyme mixture contains Taq enzyme, reverse transcriptase, UNG enzyme and RNase inhibitor.

[0113] The PCR ampli...

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Abstract

The invention discloses a combination of multiple PCR primers and probes for detecting pathogens and its application. The present invention uses influenza A virus, influenza B virus, respiratory syncytial virus, human parainfluenza virus (distinguishing type I, type II, type III), enterovirus, dengue virus, eastern equine encephalitis virus, western equine encephalitis virus Encephalitis virus, Venezuelan equine encephalitis virus, forest encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, Nipah virus, and Hendra virus were developed and designed to simultaneously detect the above pathogens Based on the combination of multiple PCR primers and probes, corresponding gene chips and detection kits were developed. The primer and probe combination designed and obtained by the invention has strong specificity, wide detection range and good result accuracy, which not only helps the prevention and control of infectious pathogens, but also can be used for inter-laboratory quality assessment of corresponding pathogens.

Description

technical field [0001] The invention belongs to the field of molecular biology technology. More specifically, it relates to a multiplex PCR primer and probe combination for detecting 18 pathogens and its application. Background technique [0002] Infectious diseases are a class of diseases caused by various pathogens that can be transmitted between humans or between humans and animals. Due to the gradual shortening of the mutation and renewal cycle of infectious diseases, the symptoms of patients are gradually aggravated, which brings a great threat to health and seriously affects the quality of life and quality of life of patients. Therefore, timely and accurate detection of infectious disease pathogens can help to take timely measures to prevent the occurrence, spread and prevalence of infectious diseases. [0003] Nucleic acid detection of infectious disease pathogens is an important means to monitor the prevalence of various infectious diseases and early diagnosis of i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/6837C12N15/11
Inventor 吴钰熙李烈军卢晓丹卢敏石琳
Owner 潮州凯普生物化学有限公司
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