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Coliphage MS2 standard sample and preparing method thereof

A standard sample, Escherichia coli technology, applied in virus/phage, biochemical equipment and methods, microbial assay/inspection, etc., can solve the problem of low reverse transcriptase and Taq enzyme activity, operator error, and inability to accurately control temperature and other problems, to achieve the effect of high titer, good uniformity and stability

Inactive Publication Date: 2015-07-15
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of nucleic acid detection, the accuracy of molecular biology detection methods is easily affected by the following factors: (1) The temperature control program of the instrument is incorrect, or the temperature cannot be precisely controlled; (2) The activity of reverse transcriptase and Taq enzyme is low 1. The quality of the reaction solution is poor, and the design of primers and probes is unreasonable; (3) the amount of nucleic acid template extraction is small, the target fragment of detection is degraded, the amount of residual ethanol and phenol is too large, and the sample impurities are not removed cleanly, such as bile in stool samples. Salt, hemoglobin in the blood and urea in the urine can inhibit the PCR reaction; (4) the nucleic acid sequence of the virus is mutated; (5) the operation error of the experimental operator

Method used

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  • Coliphage MS2 standard sample and preparing method thereof
  • Coliphage MS2 standard sample and preparing method thereof
  • Coliphage MS2 standard sample and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Escherichia coli phage MS2 standard sample preparation

[0031] (1) Preparation of host bacteria

[0032] Pick out a little ATCC 15597 Escherichia coli strain with an inoculation loop and inoculate it on a TSA-YE plate for overnight culture; pick 1 loop of bacteria into 10mL LB liquid medium, and culture overnight;

[0033] (2) Preparation of a large number of phage MS2

[0034] 1. Inoculate the host bacterium culture solution prepared in (1) into LB liquid culture medium according to 0.5% (volume ratio), at 36±1°C, after 150rpm shaking culture for 4-5h, press 0.5-1% (volume ratio) Ratio will be phage (10 8 ~10 9 pfu / mL) was inoculated into the above-mentioned bacterial culture solution to ensure that the ratio of the number of phages to the number of bacteria was 0.1-2.0.

[0035] 2. After inoculation, culture the bacterial culture solution at 36±1°C, 150 rpm for 5 hours or overnight, take the culture solution and centrifuge it at 10 000 g for 10 minu...

Embodiment 2

[0048] Embodiment 2: Construction of real-time fluorescent PCR standard curve

[0049] (1) Sample material: the prepared standard sample (1.57×10 11 pfu / mL). ABI Ambion Life Technologies AM1005 AgPath-ID One-Step RT-PCR Kit. ABI 7500 FAST real-time fluorescent quantitative PCR instrument.

[0050] (2) Construction of real-time fluorescent PCR standard curve

[0051] The prepared standard sample (1.57×10 11 pfu / mL). 10-fold serial dilution to 2.5×10 9 pfu / mL, take 2.5×10 9 200 μL of pfu / mL sample was lysed at 95°C for 5 minutes, and then 10-fold serially diluted to 10×, 100×, 1000×, 10000×, the corresponding concentration of phage MS2 was 1.57×10 8 pfu / mL, 1.57×10 7 pfu / mL, 1.57×10 6 pfu / mL, 1.57×10 5 pfu / mL.

[0052] Prepare the RT-PCR master mix according to the instructions of the RT-PCR kit. Each reaction system is 25 μL, including 20 μL of the master mix and 5 μL of samples. Each dilution is repeated 3 times, and the standard sample option is selected and set on...

Embodiment 3

[0053] Embodiment 3: Carry out the detection of Norwalk virus with MS2 as process quality control standard sample

[0054] (1) Sample materials: buy 1 part of red clam, scallop, clam and oyster from the Tuandao market in Qingdao.

[0055] (2) Use tweezers, a scalpel, etc. to open the shellfish shell, separate the digestive glands (digestive glands), collect 2g±0.2 to 50mL centrifuge tubes for each shellfish, operate on ice, and cut the viscera into pieces with sterile scissors .

[0056] (3) Add 2mL proteinase K working solution and 100μl process control substance (2.5×10 8 pfu / mL phage MS2 solution), vortexed to mix.

[0057] (4) Incubate on a shaker at 37°C for 1h, 320r / min.

[0058] (5) Water bath at 60°C for 15 minutes.

[0059] (6) Centrifuge at 3000 g at 4°C for 5 min, take the supernatant and record the total volume, take 500 μL of the supernatant to extract RNA, and finally dissolve it in 100 μL of the eluate for PCR detection.

[0060] (7) Prepare the RT-PCR mast...

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Abstract

The invention discloses a coliphage MS2 standard sample. The concentration of the coliphage MS2 standard sample is 1.57*1011 pfu / mL, and the standard sample can be used as a quality control substance for the detection process when food-borne RNA viruses are detected. According to a preparing method of the coliphage MS2 standard sample, evenness and stability are tested, and the guarantee period is 12 months.

Description

Technical field: [0001] The invention belongs to the technical field of biological analysis test, clinical medical test and food-borne RNA virus detection, and in particular relates to a coliphage MS2 standard sample. Background technique: [0002] Bacteriophage MS2 belongs to the bacterial virus of non-cellular protozoa, obligately infects and parasitizes the corresponding living bacteria, and has the basic conditions as an indicator of enterovirus in water: it is not pathogenic to humans, and it is polluted by enterovirus It is ubiquitous in the aquatic environment; high concentration inoculation and field trials can be carried out; the number is higher than that of enterovirus. It is similar to enteroviruses in terms of morphological characteristics, physical and chemical properties, resistance to natural environmental conditions and disinfectants; the detection operation has the advantages of simple and fast, safe and reliable, less affected by the environment, and simpl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N7/00
CPCC12Q1/70C12Q1/686C12Q2561/113C12Q2561/101C12Q2531/113
Inventor 房保海岳志芹张晓文王群郑小龙王宫璞梁成珠徐彪
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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