Marker for early diagnosis of respiratory syncytial virus infection

A technology of syncytial virus and respiratory tract, applied in the field of molecular diagnosis, can solve the problems of cumbersome operation, non-specific staining, difficulties, etc., and achieve the effect of reducing mortality, more accurate genetic diagnosis, and timely genetic diagnosis

Inactive Publication Date: 2019-03-08
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common rapid colloidal gold method has a short detection time but low sensitivity; the detection effect of the immunofluorescence method for antigen detection is not ideal for low-concentration infection samples [Casiano-Colon, A.E., B.B.Hulbert, et al. (2003).Lack of Sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults. JClin Virol 28(2): 169-74; Rabagliati, R., M. Serri, et al. (2007). Utility of real time polymerase chain reaction in the diagnosis of respiratory syncytialvirus infection among adult patients.Rev Chilena Infectol 24(6):441-5], the virus needs to be cultured before detection, and the requirements for operators are high, and there are problems such as non-specific staining
Virus culture requires the presence of live virus to achieve, and the operation is cumbersome
[0004] Diagnosis of RSV infection by detecting serum antibodies in patients cannot provide

Method used

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  • Marker for early diagnosis of respiratory syncytial virus infection
  • Marker for early diagnosis of respiratory syncytial virus infection
  • Marker for early diagnosis of respiratory syncytial virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Screening of genes differentially expressed in patients with respiratory syncytial virus infection and normal people

[0045] 1. Case collection population

[0046] Children treated by the inpatient department of the pediatric department of the hospital (RSV infection group, 5 cases) and children with routine physical examination in the pediatric outpatient growth and development clinic (control group, 5 cases).

[0047] 2. Clinical case screening criteria

[0048] 2.1 Inclusion criteria of RSV infection group

[0049] (1) Age: 1 month to 1 year old;

[0050] (2) Past history: no RSV infection and / or wheezing attack;

[0051] (3) Clinical symptoms: cough and / or wheezing and / or shortness of breath;

[0052] (4) Physical examination: increased respiratory rate / three inspiratory depressions / wheeze or moist rales on lung auscultation;

[0053] (5) Auxiliary examination: the throat swab is positive for RSV antigen;

[0054] (6) Imaging examination: Chest X-ra...

Embodiment 2

[0103] Embodiment 2QPCR experiment verifies the genes differentially expressed in patients with respiratory syncytial virus infection and normal people

[0104] 1. Research object:

[0105] The screening criteria were the same as in Example 1, with 30 cases in each of the RSV infection group and the control group.

[0106] 2. Extraction of total RNA in blood

[0107] Step is with embodiment 1.

[0108] 3. RT-PCR

[0109] (1)RT

[0110] RT reaction system (20μl):

[0111]

[0112] RT reaction procedure:

[0113] 42°C 15min

[0114] 85℃ 5s

[0115] 4℃ ---

[0116] (2) qPCR

[0117] PCR reaction system (20μl):

[0118]

[0119] PCR reaction program:

[0120] Amplification procedure:

[0121] Stage 1 95°C 10min

[0122] Stage 2 95°C 10s

[0123] 49.1°C 10s

[0124] Repeat for 45 cycles

[0125] Three replicate wells were set for each sample, and the internal reference was GAPDH.

[0126] (3) Primers

[0127] The primer sequences of CTSD gene and GAPDH gene ...

Embodiment 3

[0141] Example 3 Western blot experiment to verify the expression products of differentially expressed genes in patients with respiratory syncytial virus infection and normal people

[0142] 1. Research object: same as embodiment 2.

[0143] 2. Mononuclear cell isolation

[0144] Take 10ml of venous blood from patients in the RSV infection group and the control group, inject it into a sterile vial containing heparin, and shake it gently immediately after capping. Add an equal volume of HBSS (NaCl 8.0g, NaCl 2 HPO 4 0.132g, KH 2 PO 4 0.06g, KCl 0.4g, phenol red 1ml, NaHCO 3 0.35g, D-glucose 1.0g, dissolved in 1000ml double distilled water), to reduce the aggregation of red blood cells. Draw 8ml of lymphocyte stratification solution into a 50ml centrifuge tube, slowly add the diluted blood along the tube wall, keep the interface clear, do not mix the two, centrifuge at 20°C 2000r / min for 30min, carefully absorb the stratification solution and plasma transfer The turbid...

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PUM

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Abstract

The invention discloses a molecular marker of a CTSD gene for early diagnosis of respiratory syncytial virus infection. The molecular marker adopts second generation sequencing and a QPCR method to find out that the expression of the CTSD gene in the blood of normal people and respiratory syncytial virus infected patients is significantly different, namely, the molecular marker judges whether a person to be test is infected with respiratory syncytial virus or not by detecting the expression of the CTSD gene in the blood. According to the correlation between the molecular marker and the respiratory syncytial virus, the invention develops a kit for diagnosing respiratory syncytial virus infection, and the kit detects the expression of the CTSD gene to diagnose respiratory syncytial virus infection. The diagnostic kit can be used for early diagnosis of diseases, and has a wide application prospect in clinic.

Description

technical field [0001] The invention belongs to the field of molecular diagnosis and relates to early diagnostic markers for respiratory syncytial virus infection, in particular to the application of the molecular marker-CTSD gene in blood in the preparation of tools for diagnosing respiratory syncytial virus infection. Background technique [0002] Respiratory syncytial virus (RSV) belongs to the Pneumovirus genus of the Paramyxoviridae family, and is a non-segmented single-stranded RNA (negative strand) virus with an envelope. It is the most common virus that causes lower respiratory tract infection in infants and young children. It invades the human body mainly through the respiratory tract and spreads through the air (droplets, dust). The virus mainly proliferates in the nasopharyngeal epithelial cells. Respiratory syncytial virus is the most important pathogen of viral pneumonia in children under 5 years old, and it is also one of the causes of sudden infant death. Chi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70G01N33/569
CPCC12Q1/701C12Q2600/158G01N33/56983
Inventor 杨承刚任静
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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