The Vircell PCR controls have been designed to be used as positive controls in conventional or real-time PCR techniques.
Vircell PCR controls contain the purified complete DNA or RNA of the specified microorganism. In this way, any pair of primers specific for the microorganism can be used for the amplification of the infectious agent. Only in the case of PARVOVIRUS B19 (MBC080), the complete DNA of the microorganism is not included. This virus control includes a plasmid of a specific gene of the microorganism and primers for the amplification to take place. In the name of the product, its character of plasmid is specified.
Our PCR controls are supplied lyophilized. It is necessary to reconstitute the controls with the solution contained in the kit. In the case of DNA controls, the required volume is 100 μl, whereas for RNA controls it is 50 μl.
The expiry date of the Vircell PCR controls is 30 months for the DNA and 24 months for the RNA from the date of manufacture; as long as the storage recommendations described in the instructions for use of the product are followed.
5. Can the Vircell PCR controls be run on different platforms such as Smart Cycler, Light Cycler or Cobas Amplicor?
Yes, the Vircell DNA/RNA controls can be used in any nucleic acid amplification platform. The performance of our controls has been proven to be maintained regardless of the type of amplification platform on which the technique is carried out. However, it is not recommended to use DNA/RNA controls on platforms that perform nucleic acid extraction.
No, no previous extraction is required since our controls contain the purified DNA or RNA of the agent to be analyzed.
The PCR controls are supplied lyophilized and, under these conditions, should be stored refrigerated (2-8ºC). Once reconstituted, DNA controls should be stored at temperatures between -5 and -40ºC, while RNA requires temperatures between -70 and -90ºC. Vircell recommends making aliquots of the material after reconstitution to avoid potential freeze/thaw problems associated with reusing the same vial on multiple occasions. The response of the controls to repeated cycles of freezing/thawing has been analyzed, and both DNA and RNA remain stable. However, we recommend users avoid the use of unnecessary cycles of freezing and thawing in order to avoid possible degradation or contamination of the controls.
As detailed in the instructions for use of the product, it is advisable to store aliquots of the controls to avoid unnecessary freeze/thaw cycles. In the case of requiring a lower concentration of control, it is possible to make dilutions and store them for a period of time. However, our recommendation is never to store dilutions with a concentration lower than 1000 copies/μl since after a prolonged storage period or after successive cycles of freezing/thawing a reduction in the number of copies has been observed. In the case of requiring a concentration lower than that established in the control manual, the necessary dilutions should be prepared just before use.
9. What if, after using a specific control, the result is somewhat high for the requirements needed?
The controls are quantified in order to make them specifically suitable for quantitative PCR. Do not hesitate to contact us if you have any questions about the quantification of PCR controls.
The quantification of our controls has been performed by real-time PCR. The amount of copies has been obtained using a specific sequence as target. In the quantification by real-time PCR a standard curve is used as a reference by comparing the amplification of a target sequence of the microorganism with a specific standard for each of the quantified references.
12. Is it a bad practice to freeze and thaw PCR controls? How many times a PCR control can be frozen and thawed without affecting the performance of the control?
It is not advisable to freeze and thaw the PCR controls repeatedly as the quality of the product may be affected, especially when the genetic material is not very concentrated (<1000 copies / μl).
The influence of freezing and thawing on product quality will depend on the type of control, RNA being more easily degradable than DNA. In any case, it is difficult to set a maximum number of freezes / thaws that guarantees the quality of the process. Therefore, it is more convenient to reconstitute the vial when it is received and prepare aliquots before freezing.
Our PCR controls are not infectious as they only contain the purified genetic material obtained from the previously inactivated microorganism. Vircell provides a document certifying this feature upon customer request.
Vircell recommends using at least 1 μl of PCR control the first time it is used. In the quantified controls, 1 μl of volume contains approximately 15000 copies of the microorganism, allowing up to 100 reactions with each vial. However, the user can make dilutions of the control and thus determine how many copies are needed to get a robust positive control in a particular PCR system. It is not advisable to use the controls at the limit of detection (from 4 to 10 copies/control).
Vircell PCR controls offer important benefits compared to other manufacturers:
• Wide range of products - more than 120 infectious agents available
• Positive controls suitable for conventional and Real Time PCR - any platform can be used
• Versatile product line - extraction and amplification controls
• Most of the controls are quantified
• Complete purified DNA or RNA - any sequence can be amplified
• Obtained from whole microorganisms
• Designed to avoid variability factors in PCR testing
• Lyophilized presentation avoids extra transport costs and improves stability
• No need of dry ice for transport
• Competitive price for clinical diagnostic use
The PCR controls that are currently in our catalog are not encapsulated, but contain the complete and purified genetic material of the microorganism ready to use in amplification reactions.
17. What are the advantages of using Vircell's ready-to-use PCR Controls vs. in-house manufactured controls?
The use of Vircell PCR controls avoids the main challenges associated to laboratories that use their own internal controls:
• Limited quantity available
• Difficulty to obtain positive samples in esoteric or less frequent parameters
• Non standardized results; lot-to-lot inconsistency
• Non-purified controls - human and other microorganisms genetic material present in the controls
• Risks associated with infectious agents
• Less stability
The use of commercial PCR controls avoids these difficulties and provides advantages in terms of simplicity, long-term storage capacity, standardization, quality and consistency. In addition, quality accreditation groups call upon laboratories in some countries to use commercial controls.