Auleon Biologicals
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Lumpy Skin Disease Virus Real-Time PCR Test Kit (Probe)
Classification：
Details
[Name]
Common name: Bovine nodular skin disease virus fluorescent PCR detection kit (probe method)
Trade name: None
英文名称：Lumpy skin disease virus Real-time PCR test (Probe)
[Function]
It is suitable for the specific detection of bovine nodular skin disease virus in animal skin lesions, skin scab, saliva, eye and nose secretions, milk, semen, feces, vectors and other samples.
[Main composition and content]
| Serial Number | Product Components | Specifications |
| 1 | LSDVPCR reaction solution (with probe) (brown tube) | 1 tube (1.2mL) |
| 2 | LSDV positive control (red cap tube) | 1 tube (100 μL) |
| 3 | LSDV positive control (green cap tube) | 1 tube (100 μL) |
| 4 | Instructions for use | 1 part |
Note: The components of different batches are not used interchangeably. The components in the kit should be fully mixed and centrifuged before use (flick or reverse mixing is recommended, and vortex oscillator mixing is not recommended). Repeated freezing and thawing should be avoided as much as possible!
[Scope of application of fluorescence PCR instrument]
ABI7500Fast, ABI7500, Q5 series, Roche series, BIO-RAD series, such as fluorescence quantitative PCR detector, etc.
1 Sample pre-treatment
① Bovine diseased material: cut 50~100mg of bovine diseased material tissue, add it into a mortar containing 500μLPBS or physiological saline, slowly grind it into homogenate, or grind it with a tissue freezing grinder, transfer the grinded homogenate to a sterilized centrifuge tube without RNase contamination, and centrifuge it at 6000rpm for 30s; Take the supernatant for later use.
② Secretions, milk and semen produced during the ulceration of saliva and nodules: directly take the sample and centrifuge at 6000rpm for 1min; Take the supernatant for later use.
2 Sample DNA extraction (sample processing area)
Take the sample after the above pretreatment, according to the regular method to extract the virus DNA, also can use commercial kit to extract the virus DNA. A negative control participated in the extraction to monitor the environment. The positive control was used directly without extraction.
3 Preparation of PCR system
Take out the kit 3.1, take out the reagents required for the experiment, melt and mix well and centrifuge instantaneously to remove the liquid attached to the tube wall;
3.2 The reaction tube or reaction plate is selected according to different PCR instruments. The total reaction volume is 25 μL. The reaction solution is prepared in the liquid preparation area and the sample is added in the sample treatment area. The test reaction system is prepared in the following table:
| Component reaction tube | Detection tube | control tube | |
| positive control | Negative Control | ||
| LSDVPCR reaction solution (including probe) | 20μL | 20μL | 20μL |
| LSDV positive control | - | 5μL | - |
| LSDV negative control | - | - | 5μL |
| Sample DNA | 5μL | - | - |
| Total volume | 25μL | 25μL | 25μL |
4 Amplification (detection zone)
Set the reaction conditions according to the following parameters:
| reaction parameters | Pre-variable temperature | 45 cycles | |
| Denaturation | Annealing/Extension | ||
| Temperature | 95°C | 95°C | 58℃ |
| Time | 3min | 10sec | 34sec |
Fluorescence signal acquisition was set at the time of annealing/extension. For the multi-channel fluorescence PCR instrument, the fluorescein FAM is selected as the signal acquisition channel, the quenching gene is selected as None, and the dye correction is selected as None.
5 Judgment of results
5.1 experimental conditions
5.1.1 Negative control: no specific amplification curve or Ct value> 45.0, or no Ct value.
5.1.2 Positive control: Ct value should be <28.0, and there is an obvious S-type amplification curve.
5.2 result judgment
5.2.1 Positive: the Ct value of the sample test result is less than or equal to 40.0, and there is an obvious S-type amplification curve, indicating that the bovine nodular skin disease virus nucleic acid is detected in the sample.
5.2.2 Negative: The Ct value of the sample test result is> 45.0 or there is no specific S-type amplification curve, indicating that the sample does not detect bovine nodular skin disease virus nucleic acid.
5.2.3 Suspicious: The Ct value of the sample test result is in the range of 40.0~45.0, and the sample shall be rechecked. If the Ct value of the repeated experiment is still in the range of 40.0~45.0 and has obvious exponential growth, it is judged as positive, otherwise it is negative.
[Note]
1 Please read the instructions of this kit carefully before the experiment and strictly follow the operation steps.
2 All reagents should be stored at the specified temperature, see the reagent label for details.
3 In order to avoid cross-contamination, please operate in a nuclease-free environment as much as possible, and partition the test process (reagent preparation area, sample preparation area, amplification area, etc.).
4 When preparing the PCR reaction system, try to avoid bubbles, and check whether the reaction tube is tightly covered before amplification to avoid leakage of the reaction solution and contamination of the instrument.
5 In order to ensure the accuracy of the experimental results, the samples should be collected as fresh as possible, transported at 2~8 ℃, and dry ice should be used for long-distance transportation.
6 All reagents should avoid repeated freezing and thawing.
[Storage conditions and period of validity]
The kit is stored at -20°C and is valid for 12 months.
[Product performance index]
The primers and probes used in the kit are consistent with those used in the national standards being submitted for review. The minimum sensitivity of the kit for testing clinical samples is 2 copies/μL.
[Specification] 50 copies/box
[Production enterprise] Chongqing Aolong Biological Products Co., Ltd.
[Address] No.5, 4th Branch Road, Banqiao Industrial Park, Rongchang District, Chongqing
[Postcode] 402460
[Technical service] 023-46763511
[Sales hotline] 023-46761801
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