详细信息

Peste des petits ruminants virus (PPRV) nucleic acid test kit

• 2026-10-20   佚名

Peste des petits ruminants virus (PPRV) nucleic acid test kit

(Fluorescent RT-PCR)

[Product Overview]

This product is a one-step qRT-PCR nucleic acid test kit designed for the auxiliary detection of PPRV nucleic acid in samples. Specific primers and a specific fluorescent probe targeting the conserved region of the PPRV N gene have been developed; the probe is labeled with the FAM fluorescent dye. When combined with the qRT-PCR reaction mix, this kit enables rapid detection of PPRV nucleic acid using a fluorescent quantitative PCR instrument.

The kit includes a positive control and a negative control. The positive control consists of a plasmid containing the amplification fragment, used to assess the validity of the assay; the negative control is used to detect the presence of contamination in the reaction system.

[Product Composition]

[Storage Conditions]

Store at-20 °C, protected from light; shelf life: 12 months.

[Applicable Instruments]

Real-time fluorescent PCR instruments, such as the ABI 7500, Bio-Rad CFX96, and Bio-Rad 96P.

[Sample Requirements]

1. Customers are required to provide their own reagents: normal saline.

2. Collected tissue samples—including nasal swabs, oral swabs, ocular swabs, as well as lymph nodes, spleen, kidney, rectum, colon, and lung—should be stored and transported at 2–8 °C and promptly submitted for laboratory testing. Alternatively, the samples may be preserved in viral transport medium and then sent to the laboratory for analysis.

Note: Samples submitted for testing must be fresh; repeated freeze-thaw cycles are strictly prohibited.

3. It is recommended to use a commercial viral nucleic acid extraction kit for RNA extraction and purification; the extracted RNA can then be directly used for detection. If samples are not analyzed immediately after extraction, they may be stored at-80 °C for later use; however, repeated freeze-thaw cycles should be avoided.

[Operating steps]

1、 Sample Processing – Viral RNA Extraction (This kit does not include sample processing reagents)

It is recommended to perform sample pre-treatment in accordance with standards such as "GB/T 27982-2011 Diagnostic Techniques for Peste des petits ruminants virus" and "NY/T 541-2016 Technical Specifications for Sampling, Preservation, and Transportation of Veterinary Diagnostic Samples," and to utilize commercial nucleic acid extraction kits to extract nucleic acids from the samples.

1). Tissue sample processing: Take approximately 1 g of tissue sample, cut it into small pieces using surgical scissors, and mix thoroughly; then take 0.05 g of the tissue sample and place it in a mortar, add 1.5 mL of normal saline, and continue grinding; once homogenized, transfer the mixture to a 1.5 mL sterile centrifuge tube, centrifuge at 8,000 g for 2 min, and collect 100 µL of the supernatant in another 1.5 mL sterile centrifuge tube.

2). Serum sample processing: Transfer 100 µL of serum sample into a 1.5 mL sterile centrifuge tube.

3). Cell culture medium sample processing: Transfer 100 µL of cell culture medium directly into a 1.5 mL sterile centrifuge tube.

4). Viral RNA was extracted using a commercial RNA extraction kit.

2、 qRT-PCR assay (this kit is a qRT-PCR assay kit)

1). Reagent Preparation (to be performed in the reagent preparation area)

Remove the reagents required for the experiment from the kit; before use, invert the kit gently and mix thoroughly to minimize bubble formation, then perform brief centrifugation prior to use.

Based on the number of required reactions (number of test samples + positive control + negative control), pipette an adequate amount of assay reagent into a sterilized 1.5 mL centrifuge tube to prepare the assay reagent mixture. For each reaction, prepare the mixture according to the proportions specified in the table below; after thorough mixing, perform instantaneous centrifugation and set aside the mixture for subsequent use.

Note: When using the one-step qRT-PCR enzyme mix, gently mix the mixture, and when aliquoting, aspirate slowly to avoid bubble formation. Immediately after use, return the mix to a refrigerator and store it at –20 °C. For other reagents, ensure they are fully dissolved before use, followed by brief centrifugation and thorough mixing, to prevent uneven salt ion concentrations from affecting experimental results.

Prepare PCR reaction tubes based on the number of test samples, positive controls, and negative controls, and add

25 μL of detection reaction mixture to each PCR reaction tube.

2). Sample addition (performed in the sample processing area)

Add 5 μL of the test sample, positive control, and negative control, respectively, to each of the reaction tubes above, then mix thoroughly; each reaction tube will thus contain a total volume of 30 µL. Secure the tube caps, perform brief centrifugation, and transfer the contents to the amplification zone.

3). Selection of fluorescence detection channels (using the ABI 7500 as an example)

Perform FAM channel detection for PPRV (Reporter: FAM, Quencher: None); set the Passive Reference to ROX.

4). Reaction procedure settings:

[Interpretation of result]

1. Experimental setup requirements

The reported fluorescence for PPRV in this product is FAM.

1). Positive control: The FAM channel exhibited a characteristic S-shaped amplification curve, and all Ct values were ≤35;

2). Negative control: No Ct value detected in the FAM channel;

The above requirements must be met simultaneously within the same experiment; otherwise, the experiment will be invalidated, and it must be repeated.

2. Judgment criteria

For suspicious results, a retest is required; if the Ct value in the retest is ≤45, the result is classified as positive; if no Ct value is obtained, the result is classified as negative.

[Additional notes]

1. The target sequence detected by this assay kit is a conserved region of PPRV. However, if the virus undergoes a genetic mutation at this target sequence, false-negative results may occur.

2. Test results depend on the collection, storage, and transportation conditions of the samples; errors in any of these stages can lead to false-negative results; conversely, cross-contamination during sample handling may result in false-positive results.

3. This product is intended for research research use only. Before use, please carefully read this kit instruction manual and strictly follow the operating procedures.

4. Personnel performing experiments who have undergone professional training must wear protective equipment and conduct their operations within a laboratory equipped with appropriate safety safeguards.

5. The reagent kit should be stored away from light to prevent degradation of the fluorescent compounds. All consumables used, including centrifuge tubes and tips, must be autoclaved and free of DNase and RNase.

6. The test samples involved in this kit shall be regarded as containing infectious substances; all operations and handling procedures must comply with the relevant requirements of the "General Biosafety Guidelines for Microbial and Biomedical Laboratories" issued by the Ministry of Health and the "Regulations on the Management of Medical Waste."

Shenzhen Lvshiyuan Biotechnology Co., Ltd 

D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China

Tel. 86-755-28438788/86-18165709090

Fax 86-755-28938800

Email: info@lsybt.com  

www.lsybt.com