Orthopox/Monkeypox Virus Nucleic Acid Detection Kit (Real-time PCR)
[Specification] 50 tests/kit
This kit is used for in vitro qualitative detection of orthopox virus universal type and monkeypox virus nucleic acid in human rash fluid, nasopharyngeal swab, oropharyngeal swab and serum samples.
Poxviridae is a double-stranded DNA virus with the largest genome (175-225 kb) in the vira, of which orthopox virus (OPV) belongs to its first subgenus. Orthopox virus includes many viruses that can cause human disease, such as variola virus (VARV), monkeypox virus (MPV), cowpox virus (CPV) and vaccinia virus (VACV) ) and so on , in which both VARV and MPV can cause
severe rash, fever and other clinical symptoms in the population, and are highly infectious . The
virus can also be transmitted from person to person, primarily through respiratory droplets during prolonged, direct face-to-face contact or through direct contact with a patient’s body fluids or contaminated objects[3-4].
The test results of this kit are not the only indicator for the diagnosis of orthopox virus infection in patients. It must be combined with the clinical characteristics of the patient and other laboratory test data to correctly determine the infection of the pathogen, and formulate a reasonable treatment plan to make the treatment safe and effective.
This kit adopts fluorescent probe real-time PCR technology, and selects the specific conservative region of orthopox virus universal type to design specific primer probes. The 5′ is labeled with FAM, and the 3′ quencher is BHQ1. Selects the specific conservative region MPV-1 gene and MPV-2 gene of the monkeypox virus to design specific primer probes. MPV-1 gene probe 5′ is labeled with ROX, 3′ quencher is BHQ2. MPV-2 gene probe 5′ is labeled with VIC, and the 3′ quencher is BHQ1. In the process of PCR amplification, specific primers and probes bind to their respective target sequences. When Taq enzyme encounters the probe bound to the target sequence, it exerts the function of 5′-end exonuclease, making the reporter and the quencher separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, a fluorescent molecule is formed every time a DNA chain is amplified, realizing the complete synchronization of the accumulation of the fluorescent signal and the formation of the PCR product. At the same time, endogenous internal control is introduced to avoid false negatives in the test.
[Storage Conditions and Shelf Life]
The kit should be stored below -18℃ protected from light. The shelf life is 12 months. The number of repeated freezing and thawing should not be more than 4 cycles. It can be stably stored for 5 days below -18℃ and protected from light during transportation.
See the packaging label for the production date, batch number and expiration date.
Applied Biosystems™ Real time PCR system 7500, ABI 7500FAST real-time PCR system, ABI QuantStudio™5 Real-time PCR system, LightCycler® 480 fluorescence quantitative PCR system, Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument, Ultrassay Archimed X4/X6 Real Time qPCR System. etc.
1.1 Rash Fluid
Select the typical or clear and fresh rash. Clean the surface twice with a sterile saline cotton swab and dipped the skin to dry, puncture the rash with a sterile needle tip, squeeze out with a sterile cotton swab and dip the rash fluid into the preservation solution immediately, and tighten the screw cap. Wipe the needle insertion site again with normal saline or sterile alcohol, and press with a sterile dry cotton swab for a while to prevent infection.
1.2 Nasopharyngeal swab
A swab with a polyester fiber tip is used to enter through the anterior nostril and proceed slowly back and forth along the base of the inferior meatus. When the top of the swab reaches the posterior wall of the nasopharyngeal cavity (with a touch of the wall), let the swab stand for a while (about 3 seconds), then gently rotate it for a round, slowly remove the swab, and put it into the tube. Break the plastic handle by hand, immerse the swab in the sampling solution, and tighten the cap. It is validated that nasopharyngeal swab samples can be preserved with normal saline or Hank’s solution preservation solution.
1.3 Oropharyngeal swab
Wipe the posterior pharyngeal wall and bilateral tonsils at the back of the uvula (palatine or uvula) with moderate force with a polyester fiber head swab. Avoid touching the tongue, take out the posterior sampling tube, and break the plastic handle at the contact part of the hand. Put the swab into the sampling solution, and tighten the cap. It is validated that oropharyngeal swab samples can be preserved with normal saline or Hank’s solution preservation solution.
Use a sterile syringe to collect 3-5mL of venous blood from the subject in a vacuum blood collection tube (preferably with a separating gel). Leave it at room temperature for no more than 4 hours, centrifuge at 3000 rpm for 10 minutes, suck the upper serum (do not suck red blood cells) and transfer it to another sterile centrifuge tube for use.