Escherichia Coli O157: H7 Nucleic Acid Detection Kit (PCR Fluorescent Probe Method/Lyophilization)
Kit Description:
Cat.No.FP103
It is used for rapid detection and screening of E. coli O157:H7 in food, feed, water samples and environmental samples.
Descriptions
It is used for rapid detection and screening of E. coli O157:H7 in food , feed, water samples and environmental samples .
[Testing principle]
According to the principle of fluorescent PCR technology, specific primers and Taqman probes are designed for the specific gene of Escherichia coli O157: H7, and detected by a fluorescent PCR instrument, so as to realize the detection of Escherichia coli O157: H7 Qualitative detection of DNA.
Kit Contents
Note: ROX channel probe is not included.
|
Components |
Specification |
Quantity |
|
Buffer A |
Tube |
1 |
|
Buffer B |
Tube |
1 |
|
Positive control |
Tube |
1 |
|
Negative control |
Tube |
1 |
Expected Usage
It is used for rapid detection and screening of E. coli O157:H7 in food , feed, water samples and environmental samples .
Storage Conditions And Expiration Date
Store at -20℃ in the dark and avoid repeated freezing and thawing.
The validity period is 12 months, and the production date is shown on the outer packaging.
Instruments And Consumables
Fluorescent quantitative PCR instrument, pipette gun and matching tips, vortex shaker, mini centrifuge.
Usage
1. Sample Processing
1.1 Sample type: This kit is suitable for food, feed, water samples and other samples suspected to be contaminated by Escherichia coli O157:H7. For deep-processed meat products, beverages and other substances containing pigments, they need to be rinsed to avoid affecting the fluorescence signal collection.
1.2 Sample processing: Refer to "GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination of Escherichia coli O157: H7 Test" for sample preparation, enrichment culture and isolation of Escherichia coli O157: H7.
- Nucleic acid extraction
Take 20 mL of enrichment solution in a 1.5 mL centrifuge tube, add 200 μL of microbial lysate (additional kit is required), vortex for 30 seconds, centrifuge briefly, and set aside.
Remarks: The extraction of nucleic acid from the lysate should be completed within 10 minutes, and cannot be stored for a long time.
3. Nucleic Acid Amplification
3.1 Turn on the fluorescent quantitative PCR instrument for use.
Buffer A and Buffer B from the kit , melt them thoroughly, and centrifuge briefly. Add 18 μL Buffer A and 2 μL Buffer B to each PCR reaction tube. Then add 5 mL each of the negative control, extracted nucleic acid, and positive control into PCR reaction tubes, cap the tubes, and centrifuge briefly.
3.3 Transfer the PCR reaction tube to a fluorescent PCR machine , and use the following procedures to perform amplification experiments : select 25 mL for the reaction system , collect fluorescence signals at 60°C for each cycle, and select FAM for the detection channel .
|
Step |
Program |
Number of cycles |
|
1 |
37℃ 5min |
1 |
|
2 |
9 5 ℃ 3min |
1 |
|
3 |
95°C 15s |
4 0 |
|
60℃ 30s (collect fluorescence) |
Guides for problems analysis
The following is an analysis of the problems that might be encountered in the extraction of viral RNA. We wish it would be helpful to your experiment. In addition, for other experimental or technical problems other than operating instructions and problem analysis, we have dedicated technical support to help you. Contact us if you need at : 028-83361257or E-mail：Tech@foregene.com。
No RNA can be extracted or the yield of nucleic acid is low
There are usually many factors that affect recovery efficiency, such as: sample RNA content, method of operation, elution volume, etc.。
Analysis of common causes：
1.Ice bath or low-temperature (4 ° C) centrifugation during operation.
Suggestion: Room temperature (15-25 ° C) operation, never ice bath and low temperature centrifuge.
2. Improper sample storage or sample storage for too long.
Suggestion: Store samples at -80 ° C or freeze in liquid nitrogen, and avoid repeated freeze-thaw use; try to use freshly collected samples for RNA extraction.
3.Insufficient sample lysis
Recommendation: Please ensure that the sample and the working solution (Linear Acrylamide) have been thoroughly mixed and incubated for 10 min at room temperature (15-25 ° C)
4.The eluent was added incorrectly
Recommendation: Make sure that RNase-Free ddH2O is added to the middle of the membrane of the purification column
5.Improper volume of anhydrous ethanol in Buffer viRW2
Suggestion: Please follow the instructions, add the correct volume of anhydrous ethanol to Buffer viRW2 and mix them well before using the kit。
6.Improper sample usage.
Suggestion: 200µl of sample per 500μl of Buffer viRL. Excessive sample volume will result in reduced RNA extraction rate.
7.Improper elution volume or incomplete elution.
Suggestion: The eluent volume of the purification column is 30-50μl; if the elution effect is not satisfactory, it is recommended to add pre-heated RNase-Free ddH2O and extend the time placing at room temperature, such as 5-10min
8.Purification column has ethanol residue after rinsing in Buffer viRW2.
Suggestion: If ethanol still remains after rinsing in Buffer viRW2 and empty-tube centrifugation for 2min, the purification column can be left at room temperature for 5min after empty-tube centrifugation to fully remove remaining ethanol.
The degradation of purified RNA molecules
The quality of the purified RNA is related to factors such as sample storage, RNase contamination, and operation.
Analysis of common causes：
1.The collected samples were not saved in time.
Suggestion: If the sample is not used in time after collection, please store it at -80 ℃ or liquid nitrogen immediately. For the extraction of RNA molecules, try to use freshly collected samples whenever possible.
2.Collected samples were freezing and thawing repeatedly.
Suggestion: Avoid repeated freezing and thawing (no more than once) during sample collection and storage, otherwise the yield of nucleic acid will decrease.
3.RNase was introduced in the operating room or no disposable gloves, masks, etc. were worn.
Suggestion: The extraction of RNA molecules experiment is best performed in a separate RNA operation room, and the experimental table is cleaned before the experiment. Wear disposable gloves and masks during the experiment to avoid RNA degradation caused by RNase introduction.
4.The reagent is contaminated by RNase during the use.
Suggestion: Replace with new Viral RNA Isolation Kit for related experiments.
5.The RNase contamination of the centrifuge tubes, pipette tips, etc. Suggestion: Make sure that the centrifuge tubes, pipette tips, and pipettes are all RNase-Free.
The purified RNA molecules affected downstream experiments
The RNA molecules purified by the purification column will affect downstream experiments if there are too much salt ions or proteins, such as: reverse transcription, Northern Blot, etc.。
1.There are remaining salt ions in the eluted RNA molecules.
Recommendation: Make sure that the correct volume of anhydrous ethanol has been added to Buffer viRW2, and wash the purification column twice according to the correct centrifugation speed on the operating instructions；If there are still salt ions remaining, you can add Buffer viRW2 to the purification column, and leave it at room temperature for 5min. Then perform centrifugation to remove salt ions contamination to the greatest extent
2.There are remaining ethanol in the eluted RNA molecules
Suggestion: once confirming that purification columns have been rinsed by Buffer viRW2, perform empty-tube centrifugation according to the centrifugal speed on the operating instructions. If there is still ethanol remaining, it can be left for 5 minutes at room temperature after an empty-tube centrifugation to remove the remaining ethanol to the greatest extent.
Instruction Manuals:
Viral RNA Isolation Kit Instruction Manual
