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CN-112609023-B - Quality control substance for detecting respiratory tract pathogen nucleic acid and preparation method thereof

CN112609023BCN 112609023 BCN112609023 BCN 112609023BCN-112609023-B

Abstract

The invention provides a quality control substance for detecting respiratory pathogen nucleic acid, which comprises any one or more of coronavirus, influenza virus, adenovirus, mycoplasma pneumoniae, streptococcus pneumoniae, rhinovirus or/and legionella pneumophila. The invention divides the full-length genome sequence of the coronavirus into 6 target fragments, and the length of each fragment is 4000-5500 bp. The quality control substance covers all detection target sequences (or targets) of coronaviruses, has wide coverage, comprehensive detection targets and no detection omission phenomenon. The quality control material disclosed by the invention contains main pathogens infecting respiratory tract, has a wide coverage range, takes a real virus sample and a slow virus sample as raw materials, and has more accurate detection targets. The coronavirus quality control substance prepared by the invention integrates a target gene sequence into a host genome through a lentiviral vector, knocks out an autonomously replicating gene in the preparation process, has the capacity of self-inactivation, and ensures that the recombinant lentivirus cannot replicate in target cells and infect other cells.

Inventors

  • LI ERHUA
  • GAO XUNIAN
  • ZHONG FENGRAN
  • WU SHUXIAN

Assignees

  • 广州邦德盛生物科技有限公司

Dates

Publication Date
20260508
Application Date
20201225

Claims (6)

  1. 1. A quality control substance for detecting respiratory pathogen nucleic acid is characterized in that respiratory pathogens of the quality control substance are coronavirus, influenza virus, respiratory syncytial virus, adenovirus, rhinovirus, mycoplasma pneumoniae, streptococcus pneumoniae and legionella pneumophila; the nucleic acid of the coronavirus is 6 nucleic acid fragments of 4000-5500 bp; The coronaviruses comprise human coronaviruses HCoV-NL63, human coronaviruses HCoV-229E, human coronaviruses HCoV-HKU1, human coronaviruses HCoV-OC43, SRSA-CoV, MERS-CoV and novel coronaviruses, and the influenza viruses comprise influenza A viruses H1N1, influenza A viruses H5N1, influenza A viruses H7N9, influenza A viruses H9N2, influenza B viruses IVB and parainfluenza virus PIV; The standard value of human coronavirus HCoV-NL63 is 1.2X10 4 copies/. Mu.L, the standard value of human coronavirus HCoV-229E is 1.9X10 4 copies/. Mu.L, the standard value of human coronavirus HCoV-HKU1 is 1.3X10 4 copies/. Mu.L, the standard value of human coronavirus HCoV-OC43 is 2.0X10 4 copies/. Mu.L, the standard value of SRSA-CoV is 2.0X10 4 copies/. Mu.L, the standard value of MERS-CoV is 1.8X10 4 copies/. Mu.L, the standard value of novel coronavirus is 1.6X10 4 copies/. Mu.L, the standard value of influenza A virus H1N1 is 1.1X10 4 copies/. Mu.L, the standard value of influenza A virus H5N1 is 1.9X10 4 copies/. Mu.L, the standard value of influenza A virus H7N9 is 1.5X10 4 copies/. Mu.L, the standard value of influenza A virus H9N2 is 1.3X10 4 copies/. Mu.L, the standard value of influenza B virus is 2.3X10 4 copies/. Mu.L, the standard value of respiratory syncytial virus is 2.2X10 4 copies/. Mu.L, the standard value of parainfluenza virus is 1.5X10 4 copies/. Mu.L, the standard value of adenovirus is 1.3X10 4 copies/. Mu.L, the standard value of mycoplasma pneumoniae is 1.9X10 4 copies/. Mu.L, the standard value of Streptococcus pneumoniae is 1.3X10 copies/. Mu.L, the standard value of rhinovirus is 2.3X10 4 copies/. Mu.L, and the standard value of Legionella pneumophila is 2.1X10 copies/. Mu. 4 copies/. Mu.L.
  2. 2. The quality control material of claim 1, further comprising a diluent, wherein the diluent comprises dimethyl sulfoxide, ethylene glycol bis (2-aminoethyl ether) tetraacetic acid, sodium dodecyl sulfate, bovine serum albumin, guanidine isothiocyanate, tris-HCl, ethylenediamine tetraacetic acid and glycerol, and the pH value of the diluent is 7.2-7.6.
  3. 3. A method for preparing the quality control material according to claim 2, comprising the steps of: S1, preparing a respiratory pathogen culture, extracting pathogen nucleic acid, wherein the respiratory pathogen comprises coronavirus, influenza virus, respiratory syncytial virus, adenovirus, mycoplasma pneumoniae, streptococcus pneumoniae, rhinovirus and Legionella pneumophila, the coronavirus comprises human coronavirus HCoV-NL63, human coronavirus HCoV-229E, human coronavirus HCoV-HKU1, human coronavirus HCoV-OC43, SRSA-CoV, MERS-CoV and novel coronavirus, and the influenza virus comprises influenza A virus H1N1, influenza A virus H5N1, influenza A virus H7N9, influenza A virus H9N2, influenza B virus IVB and parainfluenza virus PIV; S2, detecting the content of the nucleic acid obtained in the step S1 by adopting a fluorescence quantitative PCR method, and obtaining the original concentration value of each pathogen nucleic acid; S3, diluting the respiratory tract pathogen in the step S2 to a required concentration value by using the diluent in the claim 2 to obtain the quality control substance containing the respiratory tract pathogen with the standard value in the claim 1.
  4. 4. The preparation method according to claim 3, wherein the step S1 is specifically implemented by the following method: (A) Firstly, dividing the full-length genome sequence of the coronavirus into 6 fragments, respectively connecting the fragments with a lentivirus expression vector by a seamless cloning method to obtain 6 expression plasmids, and then, co-transfecting packaging cells with the expression plasmids with qualified identification sequences and the packaging vectors to obtain coronavirus liquid, wherein at the moment, the extracted coronavirus nucleic acid is respectively 6 nucleic acid fragments containing partial genome sequences; (B) Extracting nucleic acid of influenza virus, respiratory syncytial virus, adenovirus and rhinovirus by mixing and culturing influenza virus liquid, respiratory syncytial virus liquid, adenovirus liquid and rhinovirus liquid with infected host cells respectively, harvesting the virus and extracting nucleic acid thereof; (C) Extracting nucleic acid of mycoplasma pneumoniae, legionella pneumophila and streptococcus pneumoniae by inoculating mycoplasma pneumoniae, legionella pneumophila and streptococcus pneumoniae into culture medium respectively, obtaining culture solution, and extracting nucleic acid.
  5. 5. The method according to claim 4, wherein the extracted nucleic acid is extracted using a magnetic bead nucleic acid extraction kit.
  6. 6. A method of preparing as claimed in claim 3, further comprising the steps of: (1) Subpackaging the nucleic acid of the S3, and evaluating and analyzing the performance of the nucleic acid; (2) And (3) absolute quantitative detection is carried out on the quality control object in the step (S3) by adopting a microdroplet digital PCR method, and the value to be measured is determined.

Description

Quality control substance for detecting respiratory tract pathogen nucleic acid and preparation method thereof Technical Field The invention belongs to the technical field of medical examination, and particularly relates to a quality control substance for detecting respiratory tract pathogen nucleic acid and a preparation method thereof. Background Respiratory tract infections are the most common infectious diseases and are caused by respiratory tract infectious disease pathogens, mainly by infection of respiratory systems such as nasal cavities, trachea and the like. Respiratory viruses are a large class of viruses that cause acute respiratory tissue infection or other tissue organ lesions by proliferation of airway mucosal epithelial cells using the respiratory tract as an invasive portal. It is counted that more than 90% of primary acute upper respiratory infections are caused by respiratory viruses, which have a probability of up to 62% of lower respiratory infections. Research shows that some respiratory pathogens, such as middle eastern respiratory syndrome virus, avian influenza virus and the like, have high transmission speed and wide epidemic range due to the transmission characteristics of the virions transmitted through respiratory paths, have high incidence rate in people, are extremely easy to cause epidemic and outbreak, and have great social influence. Because the pathogens capable of causing respiratory tract infection are various, patients may carry more than one pathogen, clinical signs and symptoms among different viruses and among viruses and bacteria overlap each other, so that diagnosis based on the etiology of clinical manifestations is difficult, the cause of the pathogens cannot be determined, the conventional treatment is invalid, the antibiotics are abused and the respiratory tract pathogens are subjected to iatrogenic cross infection, rapid detection of the respiratory tract pathogens is beneficial to diagnosis and accurate medication, corresponding quality control objects are needed to be developed, quality control can be performed on the rapid detection process, and the accuracy and reliability of detection results are ensured. Timely and accurate diagnosis of etiology is carried out at the early stage of virus infection, which is helpful for medical staff to accurately grasp the disease condition of patients and is also an important guarantee for smooth implementation of acute infectious disease prevention and control work. The first method to detect new pathogens was the next generation sequencing technology/high throughput sequencing technology (NGS) technology, and the nucleic acid sequences of new coronaviruses were soon determined. The 3 rd month 3 of 2020, the national health committee (Wei Jianwei) issued a new coronavirus pneumonia diagnosis and treatment protocol (trial seventh edition), and the confirmed diagnosis and treatment case required one of the following etiologies or serological evidence, namely real-time fluorescent RT-PCR detection of new coronavirus nucleic acid positivity, virus gene sequencing, high homology with the known new coronaviruses, and detection of positive serum new coronavirus specific IgM antibodies and IgG antibodies. The virus nucleic acid detection has the advantages of short window period, less time consumption, high sensitivity, strong specificity and the like, and can rapidly diagnose and dynamically observe the antiviral treatment effect in the early stage of viruses. However, there is also a certain false negative rate and potential false positive result in nucleic acid detection. "false negative" at the laboratory level means that the virus content in the sample collected is above the detection limit (also known as analytical sensitivity) of the detection reagent used, but is not detected by the laboratory. To avoid "false negatives" as much as possible, laboratories need to meet (1) the choice of reliable in vitro diagnostic reagents and (2) the standard clinical nucleic acid testing (rational zoning, capable testing personnel and strict quality control systems). The digital PCR (DIGITAL PCR, DPCR) technology is a breakthrough quantitative analysis technology which has been paid attention to and rapidly developed in recent years. In the detection of IgH heavy chain mutant genes with low abundance in a complex background in Sykes of 1992, only a single template molecule is obtained in each well by using limited dilution of a sample, and the number of starting molecules is accurately determined by calculating amplified signals after PCR, so that although the concept of digital PCR is not explicitly proposed, a basic experimental procedure of digital PCR is established, and an extremely important principle in digital PCR detection, namely the presence or absence of an end signal, is determined as a quantitative method. This is the prototype of digital PCR. In 1999, vogelstein and Kinzler et al put forward the concept of digital PCR b