RT-PCR primer and probe combination for simultaneous detection of 8 arbo encephalitis viruses and kit

A RT-PCR, encephalitis virus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc. It can meet the needs of rapid high-throughput detection, ensure high sensitivity and specificity, and meet the effect of rapid detection and monitoring work.

Inactive Publication Date: 2016-11-30
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many practical problems in the field and clinical application of biochips, such as high research costs and complicated labeling techniques, which are difficult for general laboratories to bear.
There are limitations in the standardization of methods, and the equipment

Method used

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  • RT-PCR primer and probe combination for simultaneous detection of 8 arbo encephalitis viruses and kit
  • RT-PCR primer and probe combination for simultaneous detection of 8 arbo encephalitis viruses and kit
  • RT-PCR primer and probe combination for simultaneous detection of 8 arbo encephalitis viruses and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Preparation of specimens and samples: the recombinant pseudovirus containing the amplified region of interest was used as a positive quality control product for the detection of encephalitis virus; enterovirus 71 (Enterovirus 71, EV71), enterovirus 75 ( Enterovirus75, EV75), Coxsackievirus B (CBV), Poliovirus (Pliovirus, PV), Dengue virus (DENV) Ⅰ~Ⅳ, Vibrio cholerae O1 (Vibrio cholerae O1, Nucleic acid extracts of Vc O1), Vibrio cholerae O139 (ibrio cholerae O139, Vc O139), Escherichia coli O157 (E.coli O157) (all provided by Fujian Provincial Center for Disease Control and Prevention (CDC)) as specific reference materials; Use deionized water and healthy human serum as negative quality control substances to extract the RNA of the above positive quality control substances and specific reference substances, and the negative quality control substances are set aside.

[0031] The test samples are collected by clinicians according to the actual situation. Testable sample...

Embodiment 2

[0052] A RT-PCR array kit for simultaneously detecting 8 kinds of arbovirus-borne encephalitis viruses, the kit is composed of the following reagent components: one-step RT-PCR reaction buffer (magnesium ion concentration is 2.0mmol / L), SEQ ID NO: Primer and probe combinations shown in 1-24, DEPC water, 10U / μl hot start Taq enzyme, 50U / μl reverse transcriptase, 25mM dNTPs, 40U / μl RNase inhibitor, one-step enzyme diluent, Positive controls, negative controls.

[0053] In addition, the kit also includes a 96-well assay plate and an instruction manual.

[0054] The specific usage of the kit is as follows:

[0055] Assembly of 96-well assay plates. Prepare the primer and probe mixture in advance, and then add it equally to each well of the eight-strip tube (to facilitate sample addition and reduce the error between wells), and store it at -20°C for later use. Prepare eight wells for each virus on each plate, and the distribution on the plate is as follows: figure 1 . The comp...

Embodiment 3

[0066] The performance evaluation of embodiment 3 kits

[0067] Sensitivity test: Dilute the standard 10 times to form a template, and the dilution is 10 -4 ~10 -10 , corresponding to a copy number concentration of 10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 Copy / μl 7 gradients, and set a blank control (no template was added to the system, replaced by water), according to the above reaction system and reaction conditions, one-step fluorescent quantitative RT-PCR reaction was carried out, and a standard curve was drawn to detect positive The lowest detection concentration was determined as the detection sensitivity of the method.

[0068] Establishment of the standard curve: by diluting the RNA standard by 10 times, with 10 6 ~10 0 After the copy number / μl 7 dilutions were carried out by one-step fluorescent quantitative RT-PCR, the amplification curve and standard curve were obtained ( figure 2 ); take the logarithm of the starting template as the X-axis, and the Ct v...

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Abstract

The invention specifically discloses an RT-PCR primer and probe combination for simultaneous detection of 8 arbo encephalitis viruses and a kit. The primer and probe have sequences shown as SEQ ID NO:1-24. The invention is based on the primer and probe combination, combines the sensitive and reliable advantages of real-time quantitative PCR technology and the advantages of simultaneous detection of multi-gene expression amount by microarray technology, and develops an RT-PCR array kit for simultaneous detection of 8 arbo encephalitis viruses. The kit can rapidly detect 8 arbo encephalitis viruses simultaneously by one RT-PCR reaction so as to realize integration and high throughput in RT-PCR detection of multiple pathogens. The kit provided by the invention can be applied to large-scale pathogen screening, provides technical support for diagnose of infection of pathogenic encephalitis arbo virus infection and determination of infection sources, and has important practical significance to clinical prevention and control of pathogenic encephalitis.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis of diseases, in particular to a combination of RT-PCR primers and probes and a kit for simultaneously detecting eight encephalitis viruses. Background technique [0002] Nervous system damage caused by viral infection has gradually become the most common central nervous system infectious disease clinically. Multiple viral infections such as severe encephalitis virus and enterovirus can cause different degrees of neurological symptoms. More than 80% of central nervous system viral infections are caused by enteroviruses, including Coxsackie virus, ECHO virus and polio-virus. In recent years, some newly discovered encephalitis viruses, especially a series of arboviruses and zoonotic encephalitis viruses, have gradually attracted people's serious attention due to their complicated symptoms, difficult diagnosis and high mortality. Current research shows that the clinical symptoms of encephalitis viru...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 陈瑶吴英松李明
Owner SOUTHERN MEDICAL UNIVERSITY
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