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Multiplex PCR primer and probe combination for detecting 18 pathogens and application of multiplex PCR primer and probe combination

A technology of pathogens and probes, which is applied in the detection of multiple PCR primers and probes for 18 kinds of pathogens and its application fields, can solve the problems of difficult identification and determination of virus types, harsh virus culture conditions, and low positive rate of culture, and achieve convenient Rapid screening, avoiding false positive results, simple operation

Active Publication Date: 2022-04-12
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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  • Multiplex PCR primer and probe combination for detecting 18 pathogens and application of multiplex PCR primer and probe combination
  • Multiplex PCR primer and probe combination for detecting 18 pathogens and application of multiplex PCR primer and probe combination
  • Multiplex PCR primer and probe combination for detecting 18 pathogens and application of multiplex PCR primer and probe combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 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, Nucleotides of These 18 Pathogens Targeting the sequence, we downloaded the nucleotide sequences of the representative strains of the above-mentioned infectious pathogens in the NCBI database, and designed multiple PCR primers and probes that can be used to detect the above-mentioned pathogens through multiple sequence comparison and analysis, and also designed internal standards Primers, internal standard probes and chromogenic system control probes are used to monitor ...

Embodiment 2

[0090] Example 2 Gene chip and detection kit

[0091] On the basis of multiple PCR primers and probes described in Example 1, the present invention has developed a gene chip and a detection kit for detecting the above-mentioned 18 kinds of pathogens. The gene chip includes a fixed carrier and a pathogen-specific probe fixed on the fixed carrier, that is, the probe sequence 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 probe sequence, and the probe is synthesized and modified by the primer synthesis manufacturer (Shanghai Jierui Bioengineering Co., Ltd.), and the 5' and 3' ends of the probe are both amino Chemical treatment (amino group Amino), the control probe of the chromogenic system is labeled with biotin dots.

[0092] 1. Preparation of gene chip

[0093] (1) Arrangement of probes

[0094] In this embodiment, the immobilized carrier used to prepare ...

Embodiment 3

[0104] Embodiment 3 detection method of infectious disease pathogen

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

[0106] details as follows:

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

[0108] (2) RT-PCR amplification;

[0109] Use the primer pairs 1 to 15 described in the kit to perform RT-PCR amplification on the samples to be tested. The volume of the PCR reaction solution is 45 μL, the sample volume of DNA 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 luciferin. The enzyme mixture contains Taq enzyme, reverse transcriptase, UNG enzyme and RNase in...

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Abstract

The invention discloses a multiplex PCR primer and probe combination for detecting 18 pathogens and application of the multiplex PCR primer and probe combination. According to the invention, influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, and human parainfluenza virus (differentiating type I, type II, type III, type IV, type IV, type IV, type IV, type IV, type IV, type V. A multiplex PCR (Polymerase Chain Reaction) primer and probe combination which can be used for simultaneously detecting the 18 pathogens is researched and designed by taking the 18 pathogens (type III), enterovirus, dengue virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuela equine encephalitis virus, forest encephalitis virus, Saint Lewis encephalitis virus, west Nile virus, yellow fever virus, Nipah virus and Hendea virus as targets. On the basis, a corresponding gene chip and a detection kit are developed. The primer and probe scheme designed and obtained by the invention is strong in specificity, wide in detection range and good in result accuracy, not only contributes to prevention and control of infectious pathogens, but also can be used for external quality assessment and the like of corresponding pathogens.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a combination of multiplex PCR primers and probes for detecting 18 kinds of 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 update cycle of infectious diseases, the symptoms of patients are gradually aggravated, which poses 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 will 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 of monitoring the prevalence of various infectious diseases and early...

Claims

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Application Information

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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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