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Primer and kit for bacterial detection and method for detecting bacteria in cell products

A technology of bacteria detection and kit, which is applied in the direction of biochemical equipment and methods, protozoa, microorganism measurement/inspection, etc. It can solve the problems of long time consumption and influence of detection results, and achieve the effect of improving sensitivity and fast detection speed

Inactive Publication Date: 2019-04-09
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Obviously, this method takes a long time, and the test results are easily affected by the experience of the operator; and when the culture method is tested, the bacteria mixed in the cell culture process can already be observed

Method used

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  • Primer and kit for bacterial detection and method for detecting bacteria in cell products
  • Primer and kit for bacterial detection and method for detecting bacteria in cell products
  • Primer and kit for bacterial detection and method for detecting bacteria in cell products

Examples

Experimental program
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Effect test

Embodiment 1

[0036] This example provides a primer complementary to the conserved region of the bacterial 23S rDNA sequence. The primer includes an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown in SEQ ID NO.1: 5'-AYCTTCGGGGGTARAGCACTGT-3', and the sequence of the downstream primer is shown in SEQ ID NO.2: 5'-GTCAGCATTCGCACTTCTGATAYC-3' .

[0037] The upstream primer sequence and downstream primer sequence are specific universal primers screened from the conserved region of the bacterial 23S rDNA sequence, and the bacterial 23S rDNA is obtained from NCBI (http: / / www.ncbi.nlm.nih.gov / ) After the sequence was sequenced, DNAMAN software was used to analyze the sequence to find the conserved fragments among different bacterial species, and specific general primers were designed in the conserved regions of these bacteria. At the same time, Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to analyze whether the primers can amplify sequences of sim...

Embodiment 2

[0040] This embodiment provides a method for detecting bacteria in cell products, comprising the following steps:

[0041] S1, prepare the sample:

[0042] Preparation of the test sample template: transfer 1.5 mL of the cell culture solution to a 2 mL centrifuge tube, centrifuge at 10,000 rpm for 2 min, discard the supernatant, and add 10 μL of sterile deionized water to the cell pellet to resuspend.

[0043] Prepare a positive control template: Take 1 μL of E. coli liquid as a positive control template.

[0044] Prepare the negative control template: Take 1 μL of sterile deionized water as the negative control template.

[0045] Preparation of positive interfering product template: transfer 1.5 mL of cell culture solution to a 2 mL centrifuge tube, add 2 μL of gradiently diluted E. coli bacterial liquid, centrifuge at 10,000 rpm for 2 min, discard the supernatant, and add sterile deionized water to the cell pellet. hanging.

[0046] S2, PCR amplification:

[0047] The rea...

Embodiment 3

[0054] This example explores the sensitivity of the detection system for bacterial contamination using PCR amplification technology.

[0055] The Escherichia coli bacteria liquid was carried out in turn for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 power dilution, take 10 respectively -7 、10 -6 、10 -5 、10 -4 、10 -3 Spread 10 μL of the second dilution solution on the plate, culture overnight at 37°C, and count the single colonies in the culture dish the next day. The results are shown in Table 2.

[0056] Table 2 plate colony count results

[0057] E. coli diluent

Plate colony count (CFU / 10μL)

10 -7

0

10 -6

4

10 -5

35

10 -4

291

10 -3

725

[0058] Take 10 respectively -7 、10 -6 、10 -5 、10 -4 、10 -3 、10 -2 、10 -1 2 μL of the second dilution was subjected to PCR amplification according to the steps in Example 2, and a negative control was set. The amplified fragments after PCR amplificati...

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Abstract

The invention relates to a primer and a kit for bacterial detection and a method for detecting bacteria in cell products. The primer comprises an upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are complementary to a conserved region of a 23S rDNA sequence of the bacteria. According to the primer and the kit for bacterial detection, bacterial contamination detection of cell products can be conveniently completed within 3 hours with a PCR amplification technology, rapid detection is realized, and sensitivity for detecting common bacteria can also be improved.

Description

technical field [0001] The invention relates to the field of biology, in particular to a primer for bacteria detection, a kit and a method for detecting bacteria in cell products. Background technique [0002] Bacteria are a large class of ubiquitous single-celled microorganisms. Along with yeasts and molds, they constitute the most commonly encountered microbial contaminants in cell culture due to their widespread distribution, rapid growth, and small size. After the cultured cells are contaminated by bacteria, a series of pathological changes such as turbidity of the culture medium and changes in pH value will appear, until they die, which will cause the failure of the experiment and some irreparable consequences. [0003] The traditional detection method of bacterial contamination requires culturing for 14 days in a specified medium and a suitable temperature, observing and recording whether there is bacterial growth in the petri dish every day. Obviously, this method t...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N1/11
Inventor 魏君朱亚莎
Owner IREGENE THERAPEUTICS LTD
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