Molecular marker in blood serving as diagnosis index of respiratory syncytial virus infection

A technology of syncytial virus and respiratory tract, which is applied in the field of molecular marker-TYMS as a diagnostic indicator of respiratory syncytial virus infection, can solve the problems of complicated operation, non-specific staining, slow antibody production, etc., and achieves reduction of mortality and genetic diagnosis. Timely and more effective genetic diagnosis

Inactive Publication Date: 2019-02-12
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Abstract
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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 timely basis for diagnosis and treatment for clinicians
Because the production of specific antibodies in the body is slow and the titer is low, the sensitivity of detection is not high, and the immunological method of this detection reflects an indirect indicator of infection and cannot replace direct pathogenic detection
Therefore, the detection of RSV antigen has become an important way for early diagnosis of diseases, but it is difficult to diagnose RSV infection whether it is through serological methods or antigen detection, because the virus titer is low in secretions, so it is necessary to develop sensitive, specific and practical methods. The early diagnosis method provides timely and accurate pathogen detection report for the clinic, which is of great significance for clarifying the cause, controlling the spread of the disease, adopting specific and targeted treatment methods, and preventing the abuse of antibiotics

Method used

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  • Molecular marker in blood serving as diagnosis index of respiratory syncytial virus infection
  • Molecular marker in blood serving as diagnosis index of respiratory syncytial virus infection
  • Molecular marker in blood serving as diagnosis index 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] Example 2 QPCR experiment verification of 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] 52.8°C 10s

[0124] Repeat 41 cycles

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

[0126] (3) Primers

[0127] The primer sequences of TYMS gene and GAPDH gene are...

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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Abstract

The invention discloses a molecular marker in blood serving as a diagnosis index of respiratory syncytial virus infection. The molecular marker in blood is TYMS. Experiments prove that the content ofTYMS gene and TYMS protein in the blood of people infected with respiratory syncytial virus obviously increases compared with that of normal people. Due to the expression difference between TYMS geneand TYMS protein, the content increase can serve as an index for distinguishing the people infected with respiratory syncytial virus from the non-infected people. Compared with traditional virus antibody detection method and virus antigen detection method, the non-virus index in the blood is adopted for virus detection by the invention, and high-sensitivity detection can be realized.

Description

technical field [0001] The invention belongs to the field of molecular diagnosis, and relates to a diagnostic index of respiratory syncytial virus infection, in particular to molecular marker-TYMS in blood as a diagnostic index of 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. Children are generally susceptible to RSV and have a hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158
Inventor 杨承刚任静
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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