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Preparation, application and kit of coliphage M13 internal standard quality-control product

A technology of Escherichia coli and bacteriophage, which is applied in the field of pathogen diagnosis, can solve the problems of unsuitable DNA, easy contamination of plasmids, and complicated operation, and achieves the effects of easy storage, convenient transportation and high safety.

Inactive Publication Date: 2016-01-06
GUANGDONG HECIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the sampling of β-actin in the sample is unsuccessful, it will directly affect the judgment of the result. Although the housekeeping gene and the target gene are amplified non-competitively, if the concentration difference between the target gene and β-actin in the sample is more than 10 times, directly Interfering and affecting the judgment of the target detection fragment; although the plasmid DNA has good stability, the plasmid is easy to contaminate the laboratory
Therefore, it is not suitable to monitor DNA as a quality control product; pseudovirus is an ideal choice compared to the previous two, but it requires certain technical support and is cumbersome to operate

Method used

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  • Preparation, application and kit of coliphage M13 internal standard quality-control product
  • Preparation, application and kit of coliphage M13 internal standard quality-control product
  • Preparation, application and kit of coliphage M13 internal standard quality-control product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of coliphage M13 internal standard quality control product

[0036] 1. Preparation of host bacteria

[0037] Pick out a little Escherichia coli ER2738 strain with an inoculation loop and inoculate it on LB solid medium for overnight culture; pick 1 transfer loop bacteria into 10mL LB liquid medium, and cultivate overnight to obtain a culture solution containing host bacteria;

[0038] 2. Preparation of phage M13 monoclonal

[0039] Take out 0.5ml of phage, and use 10-fold gradient dilution with LB liquid medium, take 100uL of the phage dilution gradient and mix with 300uL of the culture medium containing the host bacteria prepared in step 1, let it stand for 15 minutes, and mix it with 4mL of LB at 45°C Mix the solid medium evenly, then pour it into the prepared LB solid medium (containing IPTG / Xgal), and put it into a 37°C incubator for 18-24 hours after solidification. Pick a single phage plaque into the LB liquid medium containing Escherichia...

Embodiment 2

[0065] Embodiment 2: Homogeneity test of coliphage M13 internal standard quality control product

[0066] Take 9 tubes of the internal standard quality control product of phage M13 prepared in Example 1 stored at -20°C and dilute with 200uL sample diluent, shake, mix and centrifuge, and then take 50uL each for fluorescent PCR detection. The detection system and method are the same as in Example 1, and Perform statistical analysis to evaluate the uniformity of the phage M13 internal standard quality control product, the experimental results are shown in the attached figure 2 , and the data are organized in the following table:

[0067] internal standard

1

2

3

4

5

6

7

8

9

[0068] Control

Ct value

21.91

22.79

21.99

22.04

22.86

22.90

22.70

23.10

22.76

[0069] The relative standard deviation, that is, the ...

Embodiment 3

[0076] Example 3: Stability analysis of coliphage M13 internal standard quality control product

[0077] Three tubes of the internal standard quality control product of phage M13 prepared in Example 1 stored at -20°C were taken at different time points for fluorescent PCR determination. The stability of the standard quality control product; the samples were taken every two months in the first 8 months, and the samples were taken every other month in the next 8 months, and the fluorescent PCR assay and statistical analysis were carried out. The results are shown in the table below:

[0078]

[0079]

[0080] The regression analysis of variance results of the PCR detection Ct value of coliphage M13 are shown in the following table:

[0081]

[0082] The F value is less than the F critical value, indicating that the change of the phage M13 internal standard quality control product at each sampling time point is not significantly different, and the stability of the M13 i...

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Abstract

The invention relates to preparation, an application and a kit of a coliphage M13 internal standard quality-control product. The coliphage M13 internal standard quality-control product prepared by the method serving as an internal standard quality-control substance in a fluorescence PCR nucleic acid kit of DNA pathogen can be used for accurately monitoring the whole experiment process in PCR detection of the DNA pathogen, which can be used as the standard for determining whether the experiment is successful or not. The coliphage M13 internal standard quality-control product has good stability, is easy to store (the freeze-dried storage time is more than or equal to 1 year), is convenient to transport and high in safety, and can be used for truly realizing whole-course quality detection from sample treatment to amplification by serving as the internal standard quality control product.

Description

technical field [0001] The invention relates to the technical field of pathogen diagnosis, in particular to the preparation, application and kit of an internal standard quality control product of colibacillus phage M13. Background technique [0002] With the rapid development of molecular biology technology, various molecular biology diagnostic techniques based on polymerase chain reaction have the advantages of strong specificity, high sensitivity, good repeatability, accurate quantification, convenience and quickness, etc. As the most valuable research method in the field of biomedicine, it has been widely used in the detection of clinical specimens. In the DNA virus PCR detection, there are many factors affecting the experimental process, such as random errors in the measurement operation of the experimenter, the temperature difference between the wells of the amplification instrument, the residue of the inhibitor after the nucleic acid extraction of the sample, and the c...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/70C12Q1/68C12R1/92
Inventor 崔红李哲群商兴芳吴秋保曾家琨
Owner GUANGDONG HECIN SCI INC
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