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Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit

A technology for synchronous detection of diarrhea virus, which is applied in the field of detection kits for diarrhea virus, can solve the problems of time-consuming and labor-consuming antibodies, expensive technology costs, and increased costs, achieve good sensitivity and specificity, and avoid low specificity, The effect of saving production cost

Active Publication Date: 2014-04-23
NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During light microscope examination, mucus stool, bloody stool or bloody stool may have a lot of red and white blood cells, which are more common in bacteria such as Salmonella, invasive Escherichia coli, enterohaemorrhagic Escherichia coli, Campylobacter, Yersinia and some viruses Diarrhea caused by diarrhea, etc.; loose stools, watery stools, may have a small amount or no red and white blood cells, and are more common in diarrhea caused by enterotoxigenic Escherichia coli, rotavirus, Cryptosporidium, Aeromonas, etc., with cost Low, low requirements for laboratory hardware, but there are the following disadvantages: 1) low sensitivity, often false negative; 2) low accuracy, easy to cause misdiagnosis or misdiagnosis
Disadvantages: 1) Long time-consuming: generally 3-5 days or longer; 2) Low diagnostic efficiency: only a single bacterium can be purely cultured; for some pathogens with variable phenotypic characteristics, it is difficult to distinguish with morphological experience; in addition, due to Abuse of antibiotics, the growth of bacteria is inhibited during the detection process, resulting in false negative results; 3) Huge workload: Since every bacteria in each sampling sample must be tested, the workload is very huge, especially for some cultures. Bacteria with strict environmental requirements will increase the workload and difficulty of detection
Advantages: 1) High sample throughput: using 96-well microplate, one plate can complete the detection of multiple samples; 2) More sensitive, using the characteristics of enzyme linkage, the original antigen signal can be amplified; 3) Fast: In terms of time, it is much shorter than the traditional medium microbial culture inspection method; but it also has the following disadvantages: 1) False positives are prone to occur: due to washing and antigen coating operations, or cross-reaction due to similar antigenic surface determinants, Therefore, false positives are prone to occur; 2) Time-consuming and labor-intensive: making antibodies is a very time-consuming and labor-intensive work; 3) Uncertain sensitivity: the sensitivity of the experiment depends on the quality of the antibody, and each antibody is different. Difficult to control quality; 4) Unable to detect multi-variant viruses: Viruses that mutate rapidly, such as some RNA viruses, have multiple subtypes and often mutate. Mutations cause antibodies to fail and antigens cannot be detected
However, there are also the following disadvantages: 1) Low throughput: only one pathogenic component can be detected at a time. If multiple pathogenic bacteria need to be detected, multiple PCR instruments are required to work at the same time, with low efficiency and long cycle, which will pollute large quantities of multiple pathogens. 2) The cost is relatively high: because only one pathogen can be detected at a time, when a sample needs to detect multiple pathogens at the same time, it must be tested one by one, and the cost is relatively high
Advantages: 1) High-throughput parallel detection: When a sample needs to detect multiple pathogenic bacteria at the same time, all the results can be obtained in one experiment; 2) Simple and fast operation: the whole detection can basically produce results in 4-8 hours; but There are also the following disadvantages: 1) The technology is expensive and complicated: each sample requires a chip, and the cost is more than ¥1000 / sample, which is not conducive to large-scale promotion; 2) The synthesis and immobilization of probes are more complicated, especially the production of high-density Probe array is the main rate-limiting step; 3) It cannot be accurately quantified and the repeatability is poor; 4) The sensitivity is low: the chip method requires a large amount of nucleic acid, and generally requires multiple PCR amplification first. Dimers, hairpin structures, or different Tm values ​​lead to different amplified target fragment efficiencies, which in turn affect detection sensitivity; 5) Due to the variety of chips, it is difficult to formulate a unified quality control standard
[0016] At present, there are no relevant research reports on the kits and detection methods for the simultaneous detection of thirteen diarrheal viruses based on the GeXP multiple gene expression genetic analysis system at home and abroad

Method used

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  • Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit
  • Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit
  • Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit

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specific Embodiment 1

[0036] The present invention is a kit for simultaneous detection of thirteen kinds of diarrhea viruses, including DEPC water, 5×RT buffer, reverse transcription primer (RT primer mix), RT enzyme (full name reverse transcriptase), solution X, 10× PCR buffer, PCR primers, 25mM magnesium chloride solution, DNA polymerase, positive control (DNA fragments with specific sequences, used for quality control of the entire reaction system), 5×RT buffer, 10×PCR buffer, 25mM magnesium chloride solution, DNA polymerase was ordered from sigma company. The above-mentioned reverse transcription primers include the RT amplification primers of eleven kinds of diarrhea RNA viruses and human RNA internal references in the following table 1, and the above-mentioned PCR primers include the forward and reverse PCRs of the remaining two diarrhea DNA viruses, human DNA internal references, and reaction internal references in the table. The gene sequences of the amplification primers and PCR amplificat...

specific Embodiment 2

[0059] A method for synchronously detecting thirteen diarrhea viruses of the present invention, the detected viruses include human enteric coronavirus, enteric adenovirus 40, 41, enterovirus and its subtype (Coxsackie virus A16 type) , astrovirus, rotavirus A, B, C groups, norovirus, human biechovirus, human bocavirus type 2, sapovirus, small double segmented RNA virus, etc. (see Table 1), and collected patients Samples (fecal specimens, vomit, blood, etc.), nucleic acid extraction, reverse transcription and PCR reactions using patient nucleic acid as a template, and finally separated samples by capillary electrophoresis, the specific steps are as follows:

[0060] 1. Production of kits for the simultaneous detection of thirteen kinds of diarrhea viruses based on the GeXP multiple gene expression genetic analysis system. The components included in the kit are the same as those in Example 1 above;

[0061] 2. Collect samples and extract nucleic acids

[0062] Collect isolated ...

specific Embodiment 3

[0088] Detection Kit Sensitivity and Specificity Analysis

[0089] Sensitivity analysis: After diluting the positive control according to a certain copy number ratio, it is detected by PCR amplification and capillary electrophoresis until no signal is detected. The copy number is the lowest detection line, which is the sensitivity of the kit. Sensitivity up to 40 copies / uL.

[0090] Specificity analysis: Single-plex PCR amplification is detected as a single peak of the target fragment size by capillary electrophoresis.

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Abstract

The invention discloses a kit for synchronously detecting thirteen diarrhea viruses and a detection method of the kit. The kit comprises DEPC (diethylpyrocarbonate) water, a 5*RT (reverse transcription) buffer, a reverse transcription primer, a reverse transcriptase, an X solution, a 10*PCR (polymerase chain reaction) buffer, a PCR primer, a 25mM magnesium chloride solution, a DNA (deoxyribonucleic acid) polymerase and a positive control, and is characterized in that the reverse transcription primer comprises RT amplification primers of the eleven diarrhea RNA viruses and a human RNA (ribonucleic acid) internal reference, and has a gene sequence shown as SEQ ID NO. 1-12 (sequence identifier number 1-12), and the PCR primer comprises forward and reverse PCR amplification primers of the rest two diarrhea viruses, a human DNA internal reference and a reaction internal reference, and PCR amplification primers of the eleven diarrhea RNA viruses and the human RNA internal reference, and has a gene sequence shown as SEQ ID NO. 13-32. The kit and the detection method have the advantages of high specificity, sensitivity, flux and reliability, low cost, and no false negative results.

Description

technical field [0001] The invention relates to a detection kit for diarrhea virus and a detection method thereof, in particular to a kit for synchronously detecting thirteen types of diarrhea virus based on a GeXP multiple gene expression genetic analysis system and a detection method thereof. Background technique [0002] Diarrhea is a gastrointestinal infectious disease with a high incidence and is prevalent all over the world. Its incidence is second only to upper respiratory tract infection. Viral diarrhea is a common and frequently-occurring disease in my country. The etiology is relatively complicated, especially for infants and young children. The harm is even greater, so it is urgent to establish a rapid and efficient diagnostic method for diarrhea virus. [0003] At present, the traditional detection methods of diarrhea virus mainly include the following: [0004] (1) Routine stool examination: The routine stool examination method is to estimate the most likely pat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 吴勇陈燕芬南丽黄迎彬
Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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