sina fungiReal Cryptococcosis-25T-PQ3691

Sina FungiReal Cryptococcosis
Real-Time PCR Kit
PQ3691: 25 Preps
Kit component
Description QNTY
(μl)
Storage
(°C)
PCR Master Mix 625 -20
Primer Mix 375 -20
IC* Mix (10ng/μl) 20 -20
Positive control 50 -20
*IC: Internal Control
Storage and Stability
Sina FungiReal Cryptococcosis is a ready to use kit and should be stored at -20 °C upon arrival.
Check Kit on arrival. If the packaging is damaged the kit must not be used. Additionally, all reagents contained within
the kit are shipped frozen and should arrive frozen. If reagents are not frozen upon receipt or if the tubes have been
compromised during shipment, contact SinaClon assistance.
The performance of the kit will be guaranteed until the printed expiration date, when the contents keep in an
appropriate condition.
Kit Description
Sina FungiReal Cryptococcosis real time PCR Kit is an in vitro diagnosis kit for the qualitative detection of
Cryptococcus neoformans DNA extracted from whole blood, plasma, serum and skin. The assay is compatible with
common laboratory real time PCR equipment and can process up to 100 samples from post extraction to result in
less than 2 hours. This kit is ready to use reporting format, are detailed in below:
2. The rapid, and reliable detection provided by this kit is guaranteed by quality control processes that ensures the user can have
full confidence in assay quality and reproducibility.
WARNINGS AND PRECAUTIONS:
Chemical risks
There are no hazardous materials included in the manufacture of the Sina FungiReal Cryptococcosis real time PCR Kit. The
composition of all reagents represents no specific risk to the user or to their property.
Additional chemicals and materials may be required for procedures described in these Instructions for use. Carefully read any
warnings, instructions, or Safety Data Sheets provided by the supplier and follow general safety regulations when you hand
chemicals, biohazards, or other materials.
Biological risks
Sina FungiReal Cryptococcosis real time PCR Kit involves safe material, but working with Cryptococcus is
potentially dangerous and transmissible. All personnel are responsible for reading and following all necessary health
and safety precautions.
It is very important to wear appropriate PPE at all times; a lab coat, protective gloves and safety glasses are minimum
needed for working with these materials.
Protocol:
Remove the Sina FungiReal Cryptococcosis real time PCR Kit from the freezer and allow to thaw. Quickly vortex the tubes once
defrosted. Prepare the final reaction mix as follows:
PCR Regent:
Reagent Volume
(µL)
Master Mix 2X 25
Primer/probe mix 15
Temp 10
Total 50
qPCR program:
Step Temperature (°C) Time Number of cycles Analysis mode Data collection
Pre-incubation 95 600 sec 1 None None
Denaturation 95 25 sec
40 Quantification
None
Annealing 58 70 sec Acquire
Analysis
Cryptococcus (FAM) Internal Control (HEX)
≤ 40 / Cryptococcus detected.
Undet/blank or >40 ≤ 35 Cryptococcus not detected.
Undet/blank or >40 Undet/blank or > 35 Invalid. Sample needs to be retested by repeating PCR if there is
sufficient extracted DNA.
Channel for acquisition:
No Name of
channel
Target Source
wavelength
(nm)
Detection
wavelength
(nm)
1 FAM Crypto* 470 510
2 HEX IC* 535 556
*Crypto: Cryptococcus
*IC: internal control
Additional materials required (not provided)
General equipment
• Recommended nucleic acid extraction platform
• Validated real-time thermal cycler instrument
• Class II hood / PCR station
• Vortex mixer
• Mini centrifuge
• Centrifuge capable of microplate processing
(recommended)
• Fridge (2 to 8°C) and freezers (-25°C to -15°C)
• Micropipettes for volumes of 1 to 1000 μL
Reagents
• Extraction reagents
• Negative extraction control, for example nuclease free
water (recommended);
Consumables
• Appropriate DNase / RNase free plastic-ware for PCR
preparation
• DNase / RNase free pipette tips
• Disposable gloves, powder-Free
• PCR plastic-ware compatible with the thermal cycler
of choice
Important notes: please read before starting
▪ Follow GLP rules when performing nucleic acid
extraction from biological species.
▪ Check the procedure with a known Positive
Control, if use the kit for the first time.
It is important that the background signal should be considered
while you analysis the samples and their measurement should
be excluded
Undet: Undetermined.
*: Only if included for PCR.
Tips and Suggestions
Positive and negative controls At least one positive and negative control should be included in each analysis. All control should be
treated and test in the same manner as patient samples. A negative control that yields a positive test result is indicative of a mainly
is due to cross contamination. The assay run should be repeated using a fresh aliquot of negative control material, ensuring that the
work area and equipment are properly decontaminated and extreme care is taken during PCR set-up. A false negative result is
indicative of reagent failure or sample handling error. Ensure all reagents have been stored correctly and, where applicable, expiry
date before repeating the assay run taking extreme care during PCR set-up. Expected results for the positive control are provided.
Storage temperature
Troubleshooting
Uniformity of Ct values across different tests
The clinical significance of positive results with high Ct are difficult to interpret in the absence of clinical history and
context. Positive results with low DNA load (high Ct) can be seen in the early stages of infection (before the person
becomes capable of infection transmission) or late occurrence of infection when the risk of transmission is low.
Additional DNA quality control
1- Agarose gel electrophoresis of prepared DNA is a direct method for testing DNA quality in terms of molecular
size and conformation. Depending on the expected amount, pipette 3-5 ul eluted DNA directly on a gel.
2- Photometric determination of DNA concentration and quality:
Determination of DNA concentration is done by reading UV absorbance at 260 nm. Prepared DNA should be
vortexed shortly and diluted (if needed) accordingly by 10 mM Tris-HCl or Elution buffer. Blank and dilution
buffer should be the same. A standard procedure of measuring DNA quality is the determination of the
absorption quotient (Q) of readings at A260nm and A280nm:
Q = A260nm /A280nm
Otherwise, sample needs to be retested by re-extraction or recollection from the original source.
Problem The assay is Insufficient amplification
Possible Causes
• The initial amount of template is too low.
• Thermal cycler was not at correct temperature
Diagnostic Test
• Check DNA absorbance ratio at 260 and 280 nm (260/280); Pure DNA should has a 260/280 ratio of ≥ 1.8
• Check optimal conditions for your thermal cycler
Solutions
• Increase the number of amplification cycles,
• Increase the amount of template.
• Repeat your extraction with a new sample.
• Concentrate extracted DNA.
Sina FungiReal Cryptococcosis real time PCR Kit is shipped on dry ice and
should be stored within this advised temperature range immediately on
receipt.
For a pure DNA preparation, Q lies between 1.7 and 2.0.

Kit Quality Control:
All components of this Kit are successfully tested in term of DNA purification and amplification reaction