Canine AdenovirusType 1 (CAV-1) Fluorescence Quantification Test Kit

The following are the correct steps for using the Canine Adenovirus Type 1 (CAV – 1) real – time fluorescence quantitative PCR detection kit for testing:

I. Sample Collection

1. Clinical Samples
   • Blood Samples: Use appropriate blood – collecting equipment (such as a syringe or a blood – collecting needle and tube) to collect blood from the dog’s vein. Ensure that the operation is standardized during blood collection to avoid hemolysis. After collection, transfer the blood sample to a suitable container. If testing cannot be carried out immediately, store it as required (such as refrigeration).
   • Urine Samples: Use a clean and sterile container to collect the dog’s urine. It is best to collect mid – stream urine to reduce exogenous contamination. Conduct testing as soon as possible after collection. If storage is required, it can be stored under specific conditions (such as low temperature, addition of preservatives, etc., but consider the impact on test results).
   • Eye Secretion and Nasal Secretion Samples: Gently wipe the dog’s conjunctiva or nasal cavity with a sterile swab to obtain secretions. Place the swab in a sterile tube containing an appropriate preservation solution to avoid the swab from drying out, which may affect subsequent testing.
2. Environmental Samples
   • Use a sterile swab to wipe potentially contaminated places such as the floors, walls, and cages in dog kennels and pet hospitals to obtain environmental samples. Place the swab in a sterile tube containing a preservation solution and mark the sampling location and time.

II. Sample Processing

1. Blood Sample Processing
   • If serum or plasma needs to be extracted, centrifuge the collected blood sample. Generally, centrifuge at an appropriate speed (such as 1,000 – 3,000 revolutions per minute) for a period of time (such as 5 – 10 minutes) to separate blood cells from serum or plasma. Carefully aspirate the upper layer of serum or plasma and transfer it to a new sterile tube.
2. Secretion Sample Processing
   • Eye secretion and nasal secretion swab samples may need to be fully shaken in the preservation solution to allow the viruses in the secretions to be fully released into the preservation solution. If the sample is too thick, it may need to be appropriately diluted to ensure uniform reaction in subsequent testing.
3. Environmental Sample Processing
   • For environmental swab samples, operations such as vortex shaking or repeatedly squeezing the swab in the preservation solution may be required to allow the viruses possibly attached to the swab to fully enter the preservation solution. If the sample is likely to be interfered with by many impurities, pre – treatment such as filtering or centrifuging may be required to remove impurities, but care should be taken not to lose virus particles.

III. Nucleic Acid Extraction

1. Select an Appropriate Nucleic Acid Extraction Method
   • According to laboratory conditions and the requirements of the kit, column – based nucleic acid extraction methods, magnetic bead nucleic acid extraction methods, etc. can be selected. These methods can effectively isolate the nucleic acid of Canine Adenovirus Type 1 from samples and remove impurities such as proteins and cell debris.
2. Follow the Operation of the Nucleic Acid Extraction Kit
   • Regardless of the nucleic acid extraction method, strictly follow the instructions of the corresponding nucleic acid extraction kit. Generally, it includes steps such as lysing the sample to release nucleic acids, binding nucleic acids to specific media (such as column membranes or magnetic beads), washing to remove impurities, and eluting to obtain pure nucleic acids. During the operation, pay attention to accurately adding reagents and avoid cross – contamination.

IV. Preparation of the Reaction System

1. Prepare Reagents
   • According to the instructions of the Canine Adenovirus Type 1 fluorescence quantitative detection kit, take out reagents such as reaction buffer, primers, fluorescence probes, DNA polymerase, and dNTP, and ensure that they are within the expiration date and the storage conditions meet the requirements.
2. Accurately Add Reagents
   • In a sterile and nuclease – free reaction tube, accurately add the reaction buffer, primers, fluorescence probes, DNA polymerase, dNTP, and extracted nucleic acid samples (or control samples) according to the specified volume. Generally, first add the reaction buffer, then add other reagents one by one, and finally add the nucleic acid sample. When using a pipette, ensure accurate operation and avoid generating bubbles.

V. PCR Reaction

1. Set PCR Instrument Parameters
   • Place the prepared reaction system into a real – time fluorescence quantitative PCR instrument and set the PCR reaction program according to the instructions of the kit. Parameters usually include pre – denaturation temperature (such as 94 – 95°C) and time (such as 3 – 5 minutes), denaturation temperature (such as 94 – 95°C) and time (such as 15 – 30 seconds), annealing temperature (determined according to primer design, such as 50 – 65°C) and time (such as 15 – 30 seconds), extension temperature (such as 72°C) and time (such as 30 – 60 seconds), and the number of cycles (such as 30 – 40 cycles), etc. There may also be a post – extension holding stage at the end (such as holding at 72°C for 5 – 10 minutes).
2. Start the PCR Reaction
   • After confirming that the parameter settings are correct, start the fluorescence quantitative PCR instrument for the reaction. During the reaction process, the instrument will automatically monitor and record the change in fluorescence signal for each cycle.

VI. Result Analysis

1. View Fluorescence Curves
   • After the PCR reaction is completed, view the curve of the change in fluorescence signal intensity with the number of cycles through the software of the real – time fluorescence quantitative PCR instrument. Observe the shape of the curve, the time and intensity of the appearance of the fluorescence signal, and other characteristics.
2. Judge Results
   • Positive Results: If the fluorescence curve of the sample shows a significant increase in fluorescence signal within an appropriate number of cycles and exceeds the set threshold, and the positive control reacts normally and the negative control has no signal, then the sample is judged to be positive, indicating the presence of Canine Adenovirus Type 1 nucleic acid.
   • Negative Results: If the fluorescence curve of the sample does not show a significant increase in fluorescence signal, and the negative control is normal and the positive control has a signal, then the sample is judged to be negative, that is, no Canine Adenovirus Type 1 nucleic acid is detected.
   • Invalid Results: If the positive control does not show the expected fluorescence signal, or the negative control shows a fluorescence signal, or the instrument shows a fault prompt, etc., then the test result is invalid and needs to be retested.

Throughout the entire testing process, strictly adhere to the principle of aseptic operation, avoid cross – contamination between samples and reagents, and ensure the accuracy of the operation steps to guarantee the reliability of the test results.