precautions for using the Canine Parainfluenza Virus (CPIV) quantitative fluorescence detection kit

The following are some precautions for using the Canine Parainfluenza Virus (CPIV) quantitative fluorescence detection kit:

I. Sample Collection and Processing

1. Sample Collection
   • Selection of Sample Types
     • Nasal swabs and throat swabs are commonly used sample types. However, when collecting, ensure that the swabs reach the appropriate depth. For nasal swabs, gently rotate and insert them into the nasal cavity to a certain depth, avoiding excessive stimulation that may cause discomfort to the dog or damage the nasal mucosa. For throat swabs, accurately wipe the throat mucosa to prevent only collecting oral saliva, which may affect the test results.
     • When collecting blood samples, pay attention to selecting the appropriate venous puncture site and using the appropriate – sized blood collection tubes to ensure smooth collection of blood samples and avoid hemolysis, as hemolysis may interfere with subsequent nucleic acid extraction and detection.
   • Collection Tools and Operating Procedures
     • The swabs and blood collection tubes used must be sterile to prevent exogenous microorganisms from contaminating the samples. During the operation, the operator should wear sterile gloves to avoid bacterial contamination of the samples from hands.
     • After sample collection, place the samples into appropriate preservation fluids or containers as soon as possible and label them properly to prevent sample confusion.
2. Sample Processing
   • When processing samples in the laboratory, strictly follow the instructions of the kit. The sample – processing environment should be kept clean and sterile, and it is best to operate in a biological safety cabinet to prevent sample contamination during processing.
   • Follow the specified operating procedures for nucleic acid extraction, such as accurately adding the amounts of lysis buffer, nucleic acid extraction reagents, etc., to avoid improper reagent amounts affecting the purity and yield of nucleic acid extraction.

II. Reagent Storage and Use

1. Storage Conditions
   • Various reagents in the kit should be stored according to the temperature and environment required by the instructions. Generally, unopened reagents should be stored at – 20 °C or the specified low – temperature environment to prevent loss of reagent activity.
   • The fluorescent probes and Taq DNA polymerase in the quantitative fluorescence PCR reagents are relatively sensitive to temperature. Special attention should be paid to avoiding repeated freezing and thawing during storage and retrieval. They can be divided into small aliquots for storage, and one aliquot is used each time to reduce the impact of freezing and thawing on reagent activity.
2. Pre – use Inspection
   • Before using the kit, check whether each reagent is within the expiration date and whether the packaging is intact. If any abnormal phenomena such as leakage, discoloration, or precipitation of the reagents are found, stop using them.
   • Confirm that the positive and negative controls are in normal state. The positive control should stably produce the expected positive results, and the negative control should always remain negative. If the controls are abnormal, it may indicate that the kit has failed or been contaminated.

III. Experimental Operation

1. Sample – Adding Operation
   • When using a pipette to aspirate samples and reagents, ensure the accuracy and precision of the pipette and calibrate it regularly. During the sample – adding process, prevent the pipette tips from touching other non – target containers to avoid cross – contamination.
   • The sample – adding volume should be accurate. Strictly add samples, primers, enzymes, buffers, and other reagents according to the volumes specified in the kit instructions. Inaccurate sample – adding may lead to PCR reaction failure or inaccurate results.
2. PCR Reaction Operation
   • Place the PCR reaction tubes in an appropriate quantitative fluorescence PCR instrument and ensure that the instrument parameters are set correctly, such as denaturation temperature, annealing temperature, extension temperature, and cycle number. Different kits may have specific requirements for these parameters, so check them carefully.
   • Avoid frequently opening and closing the door of the PCR instrument during the PCR reaction to prevent temperature fluctuations from affecting the reaction results.

IV. Result Interpretation

1. Data Analysis
   • The interpretation of quantitative fluorescence PCR results should be based on scientific and reasonable standards. Pay attention to analyzing the growth curves of fluorescence signals and Ct values. Different sample types and testing purposes may have different interpretation thresholds.
   • For samples with results near the critical value, it is recommended to repeat the test to ensure the accuracy of the results.
2. Result Recording and Reporting
   • Accurately record the test results of each sample, including sample number, test date, Ct value, result judgment (positive or negative), etc., to ensure the traceability of the results.
   • When issuing test reports, strictly follow the laboratory specifications and relevant standards. The report content should clearly and accurately reflect the testing situation.