Monkeypox virus real Time PCR detection (100 tests)

Monkeypox virus real Time PCR detection (100 tests)

Cat. No.: BMG-QP2180-01
Size: 100 tests


Description

The Monkeypox Virus Detection Kit is a qualitative in vitro real time PCR test for the detection of DNA extracted from lesion material. DNA can be extracted from acceptable specimen types including, but not limited to, lesion fluid on a dry swab, lesion fluid swab in viral transport media, lesion fluid on a slide, crust, or lesion roof. The clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or coinfection with other viruses. The agent detected may not be the definite cause of disease. Negative results do not preclude monkeypox virus infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.


Principle

The purpose of this protocol is to describe the procedure used for the detection of Monkeypox virus DNA in clinical specimens by real-time PCR. This assay detects DNA at varying concentrations, providing a qualitative result of either positive, negative, or inconclusive in the identification of Monkeypox virus infections. Primer and probe are designed to detect Monkeypox genes. The assay kit also contains a positive control (Plasmid DNA) and a negative control for testing process monitoring.


Reagents and materials provided
 

Item	Component	Quantity		Volume /T
		100 Tests / Box	500 Tests / Box
1	MPXV PCR Mix	1.5 mL each	7.5 mL each	14 µL
2	Enzyme Mix	110 μL	550 μL	1 µL
3	Positive Control	50 μL/ tube × 1	250 μL/ tube × 1	5 μL
4	Negative Control	50 μL/ tube × 1	250 μL/ tube × 1	5 μL

Storage instructions

Store the kit frozen at -20±5°C and avoid repeated freeze-thaw cycles (less than 5 frozen-thaw times allowed). The product is valid for 12 months, and the expiry date of the product is shown on the label.

Specimen type and preservation

Specimen type: oropharyngeal swabs, nasopharyngeal swabs and sputum.
Specimen preservation: process specimens for viral RNA extraction and nucleic acid detection as soon as possible. Specimens can be stored at 4°C for 24 hours, or frozen at -80°C for longer storage, if not processed immediately. Avoid repeated freeze-thaw cycles during transport and storage of the specimens

Assay PCR reaction setup (at reagent preparation area)

A. Reagent Preparation
To calculate the number (n) of PCR reaction tubes (n= number of specimens to be tested + number of positive controls + number of negative controls), add n x14 μL of SARS-CoV-2 PCR Mix and n x 1 μL of RT-PCR Enzyme Mix to a clean tube, vortex to mix well, centrifuge and aliquot 15 µL of the mix into each PCR reaction tube.

B. Addition of Viral RNA
Add 5 μL of viral RNA extracts of the specimen or positive/negative control to each PCR tube according to the reaction preparation table below for testing. Then immediately cap the tube tightly, briefly spin the mixture and move it to the amplification area for RT-PCR detection

• PCR Reaction Preparation

C. RT-PCR amplification (at Amplification Area, use Applied Biosystems 7500 Real-Time PCR System as an example)

1. 1. Place the complete PCR reaction tubes into the fluorescent quantitative PCR analyzer and label positive control, negative control and testing specimen on the Thermocycler software.
2. 2. Enter the PCR reaction volume: 20 μL.
3. 3. Select fluorescence detection channels: JOE, FAM and Cy5 channels. Cy5 is the E gene indicator channel, FAM is the ORF1ab indicator channel, and JOE is the RNase P gene indicator channel (internal control).
4. 4. Select PCR cycle parameter setting following the instructions below, save the file and start the PCR run.
5. 5. Analyze the results according to your thermocycler manufacturer’s instructions. If the positive and negative control samples do not meet their respective requirements, the PCR run must be repeated.

Determination of results

1. The results can only be determined when the results of both controls meet their requirements.
2. Criterion for a positive specimen: a specimen can be reported as positive when FAM and JOE are both positive.
3. Criterion for a negative specimen: a specimen can be reported as negative when the JOE channel is positive, and the FAM channel is negative.
4. Cut off values must be determined by each laboratory during the validation phase.

Compatibility of real-time PCR thermocyclers
Product performance

Product analysis performance evaluation results:

1. 1. The analytical sensitivity of this kit is 250 copies/mL.
2. 2. Cross-reaction: no cross-reaction with other pathogens.