Preparation and application of coliphage MS2 internal standard quality control product and kit

A technology of Escherichia coli and bacteriophage, which is applied in the field of pathogen diagnosis, can solve the problems of easy contamination of the laboratory by plasmids, cumbersome operation, unsuitable monitoring, etc., and achieves the effects of easy in vitro culture, high safety, and convenient transportation.

Inactive Publication Date: 2016-01-13
广东和信健康科技有限公司
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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 pl

Method used

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  • Preparation and application of coliphage MS2 internal standard quality control product and kit
  • Preparation and application of coliphage MS2 internal standard quality control product and kit
  • Preparation and application of coliphage MS2 internal standard quality control product and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: Preparation of coliphage MS2 internal standard quality control product

[0046] 1. Preparation of host bacteria

[0047] Use an inoculation loop to pick out a little Escherichia coli ATCC15597 strain and inoculate it on #271 solid medium for overnight culture; pick 1 loop of bacteria into 10mL #271 liquid medium, and cultivate overnight to obtain a culture solution containing host bacteria;

[0048] 2. Preparation of monoclonal phage MS2

[0049] Take out 0.5ml of phage, and use #271 liquid medium for 10-fold serial dilution, take 100uL of the serially diluted phage liquid and mix with 300uL of the culture medium containing the host bacteria prepared in step 1, let it stand for 15 minutes, and mix with 4mL of 45℃ Mix the #271 semi-solid medium, then pour it into the prepared #271 solid medium, and put it into a 37°C incubator for 18-24 hours after solidification. Pick a single phage plaque into the liquid medium containing Escherichia coli cultured by th...

Embodiment 2

[0075] Embodiment 2: Homogeneity test of coliphage MS2 internal standard quality control product

[0076] Take 9 tubes of standard bacteriophage MS2 prepared in Example 1 stored at -20°C and dilute with 200uL sample diluent, shake, mix and centrifuge, take 50uL each for fluorescent RT-PCR detection, the detection system and method are the same as in Example 1, and carry out Statistical analysis, to evaluate the uniformity of phage MS2 internal standard quality control, the experimental results are shown in the appendix figure 2 , and the data are organized in the following table:

[0077] freeze-dried product

1

2

3

4

5

6

7

8

9

Ct value

17.94

18.19

18.02

18.04

17.99

18.0

18.28

18.64

18.01

[0078] The relative standard deviation, that is, the coefficient of variation, is used to evaluate the uniformity, which is represented by CV:

[0079] Average value X=(Ct1+Ct2+Ct3+····+Ct9) / 9 ...

Embodiment 3

[0085] Example 3: Stability analysis of coliphage MS2 internal standard quality control product

[0086] Take 3 tubes of phage MS2 internal standard quality control prepared in Example 1 stored at -20°C at different time points for fluorescence RT-PCR determination, the detection system and method are the same as in Example 1, and perform statistical analysis of phage MS2 at each sampling time point The stability of the internal standard quality control product; samples were taken every two months in the first 8 months, and samples were taken every other month in the next 8 months, and the fluorescent RT-PCR assay was carried out for statistical analysis. The results are shown in the table below:

[0087]

[0088] The regression analysis of variance results of the RT-PCR detection Ct value of coliphage MS2 are shown in the following table:

[0089]

[0090] The F value is less than the F critical value, indicating that the change of the phage MS2 internal standard quali...

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Abstract

The invention relates to a preparation method and application of a coliphage MS2 internal standard quality control product and a kit. The coliphage MS2 internal standard quality control product prepared by the method, as an internal standard quality control material of a kit for RNA pathogen fluorescent RT-PCR nucleic acid detection, can be used for accurately monitoring an entire experiment process in the RNA pathogen RT-PCR detection, so that a standard for judging whether the experiment is successful or not is provided. The coliphage MS2 internal standard quality control product prepared by the method is good in stability, easy to be preserved (more than or equal to 1 year under a state of being freeze-dried), convenient to transport and high in safety; and the coliphage MS2 internal standard quality control product, as an internal standard quality control product, can really achieve whole-process quality detection from sample processing to amplification.

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 coliphage MS2. Background technique [0002] With the rapid development of molecular biology technology, various molecular biology diagnostic techniques based on polymerase chain reaction, because of their strong specificity, high sensitivity, good repeatability, accurate quantification, convenient and fast, etc. The most valuable research tool in the field of biomedicine has been widely used in the detection of clinical specimens. In the RT-PCR detection of RNA virus, there are many factors that affect 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 target to be amplified...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/70C12Q1/68
Inventor 崔红李哲群商兴芳吴秋保曾家琨
Owner 广东和信健康科技有限公司
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