Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

A kit for simultaneous detection of eighteen kinds of pathogens with fever and rash and its detection method

A technology for simultaneous detection of pathogens, applied in the direction of microbial-based methods, biochemical equipment and methods, and microbial measurement/inspection, which can solve the problems of time-consuming and labor-consuming antibodies, expensive technical costs, and increased costs, and achieve good sensitivity and specificity, avoid low specificity, and save production costs

Active Publication Date: 2014-10-01
NINGBO HEALTH GENE TECHNOLOGIES CO LTD
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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
However, there are the following disadvantages: 1) False positives are prone to occur: due to the operation of washing and antigen coating, or cross-reaction due to similar antigenic surface determinants, false positives are prone to occur; 2) Time-consuming and labor-intensive: making antibodies is very expensive Time-consuming and labor-intensive work; 3) Uncertain sensitivity: the sensitivity of the experiment depends on the quality of the antibody, and the quality of each antibody is different, and the quality is difficult to control; 4) Unable to detect multi-variant viruses: the variability is faster Some viruses, such as influenza A virus, have multiple subtypes and often mutate, which leads to the failure of antibodies and the inability to detect antigens
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
[0015] At present, there are no relevant research reports on the kits and detection methods for the simultaneous detection of 18 kinds of pathogens with fever and rash based on the GeXP multiple gene expression genetic analysis system at home and abroad

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kit for simultaneous detection of eighteen kinds of pathogens with fever and rash and its detection method
  • A kit for simultaneous detection of eighteen kinds of pathogens with fever and rash and its detection method
  • A kit for simultaneous detection of eighteen kinds of pathogens with fever and rash and its detection method

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0035] The present invention is a kit for synchronous detection of eighteen kinds of pathogens with fever and rash, including DEPC water, 5×RT buffer, reverse transcription primer (RT primer mix), RT enzyme (full name reverse transcriptase), X solution , 10×PCR buffer, PCR primers, 25mM magnesium chloride solution, DNA polymerase, and the positive control substance is obtained by cloning each target pathogen, including the target fragment plasmid. 5×RT buffer, 10×PCR buffer, 25mM magnesium chloride solution, and DNA polymerase were ordered from Sigma Company. The reverse transcription primers include the RT amplification primers of the five fever and rash pathogens in Table 1 and the RT amplification primers of the human RNA internal reference. The above PCR primers include the positive and negative of the remaining thirteen fever and rash pathogens in Table 1. PCR amplification primers, forward and reverse PCR amplification primers of herpes simplex virus 1,2 universal type, ...

specific Embodiment 2

[0064] The present invention is a method for synchronously detecting eighteen kinds of pathogens with fever and rash. The detected pathogens with fever and rash include herpes simplex virus 1, herpes simplex virus type 2, varicella-zoster virus, African lymphoma virus, Human herpes 6 virus, human herpes 7 virus, enterovirus and its subtype (Coxsackievirus A16), measles virus, rubella virus, parvovirus B19, foot-and-mouth disease virus, Streptococcus pyogenes, Mycoplasma pneumoniae, dengue virus , typhoid, paratyphoid Salmonella, Borrelia burgdorferi, etc. (see Table 1), collect patient samples (nasopharyngeal swab, sputum, herpes fluid, blood, etc.), extract nucleic acid, and use patient nucleic acid as a template for reverse transcription React with PCR, and finally separate the sample by capillary electrophoresis, the specific steps are as follows:

[0065] 1. Production of kits for simultaneous detection of eighteen kinds of pathogens with fever and rash based on the GeXP m...

specific Embodiment 3

[0093] Detection Kit Sensitivity and Specificity Analysis

[0094] Sensitivity analysis: After diluting the positive control according to a certain copy number ratio, PCR amplification and capillary electrophoresis detection until no signal is detected, the copy number is the lowest detection line, which is the sensitivity of the kit. The detection sensitivity is up to 40 copies / uL.

[0095] Specificity analysis: Single-plex PCR amplification is detected as a single peak of the target fragment size by capillary electrophoresis.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a kit capable of synchronously detecting eighteen kinds of fever with eruption pathogens and a detection method thereof. The kit comprises diethylpyrocarbonate (DEPC) water, 5*reverse transcriptase (RT) buffer solution, reverse transcription primers, reverse transcriptase, X solution, 10* polymerase chain reaction (PCR) buffer solution, PCR primers, 25m M magnesium chloride solution, deoxyribonucleic acid (DNA) polymerase and positive reference substances. The kit is characterized in that the reverse transcription primers comprise five kinds of fever with eruption pathogens and ribonucleic acid (RNA) internal reference RT primers, and the sequences are shown as SEQ ID NO.1-NO.6. The PCR primers comprises the remaining thirteen kinds of fever with eruption pathogens, herpes simplex virus 1, 2 universal type, human DNA internal reference, forward and reverse PCR amplification primers of a reaction internal reference, and the five kinds of fever with eruption pathogens and PCR amplification primers of a human RNA internal reference. The gene sequence is shown as SEQ ID NO.7-NO.44. The kit has the advantages of being strong in specificity, high in sensitivity, high in flux, strong in reliability, low in cost, and free from false-negative results.

Description

technical field [0001] The invention relates to a detection kit and a detection method for pathogens with fever and rash, in particular to a kit for synchronously detecting eighteen kinds of pathogens with fever and rash based on a GeXP multiple gene expression genetic analysis system and a detection method thereof. Background technique [0002] Fever with rash is a disease with fever and symptoms as the main clinical manifestations, including measles, rubella and other RFIs, and often occurs in the form of outbreaks. The existing fever with rash disease has a high incidence rate, especially for infants and young children, and poses a huge threat to human health and social development. The clinical symptoms of such diseases are generally mild, but severe complications and their histological similarities make accurate diagnosis of pathogens and dialectical treatment critical. At present, most methods of diagnosis of fever with rash, such as pathogen culture, serological diag...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/14C12Q1/04C12R1/93C12R1/35C12R1/46C12R1/42C12R1/01
Inventor 吴勇陈燕芬南丽黄迎彬
Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products