DiAGSure® Human Metapneumovirus (HMPV) Detection Kit

Kit Contents

GSure Plasmid DNA Isolation Kit is an alkaline lysis based plasmid DNA isolation kit which employs silica membrane mini spin column for rapid and fast purification of the isolated DNA molecule. This kit can isolate covalently closed circular (ccc) plasmid DNA molecule from fresh overnight grown E.coli cells. Isolated plasmid is free from genomic DNA/Protein or RNA contamination. Isolation of plasmid DNA completes within 30 min. The kit can isolate at least 3μg plasmid DNA from 1ml of overnight grown culture of high copy number plasmid. For low copy number plasmid, yield generally varies from 500ng-1.5μg from 1ml culture. Yield could also be enhanced by chloramphenicol. GSure Plasmid DNA Isolation Kit can isolate plasmid DNA from 1ml to 12 ml of overnight grown cells. More than 75% of the isolated plasmid DNA is in ccc form which is the best suitable form of plasmid DNA molecule for any sort of downstream application. Most ccc form of plasmid DNA found upto 3ml of starting culture volume. More than 3ml culture does not inhibit plasmid DNA isolation, but the amount of ccc form of DNA decreases.

GSure Plasmid DNA Isolation Kit contains 3 buffers which assure complete lysis of cells and digestion of contaminating proteins. Wash buffer supplied with the kit confirms complete removal of proteins and contaminants from the silica membrane. Optimized protocol guaranties extremely purified plasmid DNA isolation reproducibly. All the buffers should be stored at room temperature. Prescribed volume of absolute ethanol is to be added with the wash buffer before starting the work.

Quality Control System
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Specification

#Operation procedure

A. Sample solution preparation:
Clinical samples are extracted according to the corresponding requirements and steps
with the viral RNA extraction kit, and the extracted RNA could be directly used for
detection. If the extracted RNA is not immediately tested after extraction, they can also
be stored at -80°C and repeated freezing and thawing should be avoided. It is advised
to start amplification with at RNA of high purity (O.D.260/280 = 2.0). A starting RNA
concentration of at least 10 ng/ µL should be used. Too high RNA concentration might
necessitate template dilution according to requirement.

B. Preparation of amplification reagent:
Take the reagents out from the -20˚C freezer and thaw them on ice. After thawing, briefly vortex the reagents followed by a short spin. The PCR reagents should be kept on ice.

C. TaqMan qRT-PCR protocol:
Per sample, set up a single reaction according to the following table:

D. Real-time PCR Instrument set up:
Set up the PCR tubes/strips/plate in the Real time PCR machine and label the sample slots accordingly. Select the sample type (Unknown/PC/NTC) and select the acquisition channel for each slot as follows:

E. PCR cycling conditions:

G. Performance Evaluation:

• I. Specificity:
  The target sequences detected in this kit specifically targets the nucleoprotein (N) gene of HMPV. The primers used in the assay failed to give any amplification from RNA extracted from the sputum of healthy individuals eliminating the possibility of non-specific annealing with control human RNA.
  Furthermore, the primers did not cross-react with other viruses like COVID-19, RSV, Rhinoviruses, Influenza virus A & B, Adenovirus, Parechovirus, Bocavirus, Mumps virus, Dengue virus, Chikungunya virus, Hepatitis C virus and Hepatitis B virus. Also, the primers did not cross-react with the most common bacteria like E. coli, B. subtilis, Pseudomonas sp. as well as pathogens like Mycobacterium tuberculosis, Streptococcus pneumoniae, Vibrio cholerae, Chlamydia trachomatis, and Salmonella typhi, and the protozoa Plasmodium sp. and Giardia lamblia.
• II. Detection limit:
  The absolute sensitivities were obtained using a dilution series of quantified synthetic RNA (in-vitro transcribed) as a template for detection. The Limit of Detection (LoD) was found to be 50 RNA copies/reaction under in-vitro conditions.
• III. Safety information:
  The DiAGSure^® Human Metapneumovirus (HMPV) Detection Kit is for laboratory use only. Use proper safety measures while handling clinical samples, like wearing mask, gloves, lab-coat, etc.
• IV. C_t cut-off for each fluorescent channel:
  
  

  Target	Cut-off Ct	Interpretation
  Viral gene – nucleoprotein (N) gene	Ct≤40	Viral RNA positive
  IC (Cy5)	Ct≤35	Internal control positive

   

#Interpretation of the results:

Assessment of clinical specimen test results should be performed after the positive and negative controls have been examined and determined to be valid. If the controls are not valid, the patient’s result cannot be interpreted.
Refer to the table below for the validity and the interpretation of each specimen result according to the results of each channel.

* If the target gene signal (FAM Channel) is strong, the IC (Cy5 Channel) may be negative.

#General precautions

Performing lab activities should always be done in compliance with general safety regulations. For more information on chemicals, consult the appropriate material safety data sheets (MSDS), which is part of the kit insert.
The following precautions should be taken to both avoid contamination and allow optimal performance and reproducibility of the assays:
• The PCR assay should only be performed by qualified laboratory personnel.
• When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
• Physically separate the workplaces as outlined in table 3.
• Use disposable tips containing hydrophobic filters to prevent cross-contamination.
• Use DNase-free PCR vials.
• Thaw DNA samples always on ice and keep them on ice or on a cooling block.
• Keep enzymes always on ice or on a cooling block when taken out of the freezer. Handle enzymes with care and mix very gently.
• When thawed, spin down the reagents for 5 seconds in a centrifuge and mix by gently pipetting up and down.
• The cycling program should be programmed in the real-time PCR instrument before performing the assay.
• Spin down the PCR mixtures shortly in the PCR plate before transferring to the real- time cycler (not necessary for RGQ).
• Do not open the PCR vials/plates after PCR amplification.