News November 10 News November 09 Cayman Chemical Chemical Probes. News November 04 News November 02 Meet our new supplier Atlas Antibodies. News October 28 Custom Solutions: Cloning to antibody development. Custom Services Origene. News October 26 Subscribe to our newsletter Get the latest news within your area of research to your inbox. Our division deals with the diagnostics of various highly pathogenic viruses, with real-time PCR as the most popular approach. For those viruses, for which there are no commercially available kits and also for confirmation of positive results obtained by using kits, we apply in-house tests using a basic one-step RT-PCR reaction mix from a manufacturer A.
These assays are thoroughly validated with this reaction mix. According to quality management rules, we always compare a new batch of the basic reaction mix with the old one. Since there are currently about 40 validated in-house RT-PCR assays in our portfolio, not every single assay is subjected to comparison, but some in-house assays are selected exemplarily and compared. Usually these requests are urgent, due to the public health precautions potentially required for highly contagious patients.
Both assays were set up in parallel using the same aliquot of water and reaction mix and were run in parallel on the same Applied Biosystems Real-Time PCR Instruments. The sample was negative for YFV with perfectly fine amplification curves for the positive control in vitro transcribed RNA. In contrast, the LASV assay did not show any amplification, neither for the sample nor for the positive control. Even on an agarose gel no amplification of the positive control could be shown.
Since these results could not be explained easily, we started troubleshooting by checking 12 parameters [3] , which were sometimes combined in one run. Table 1 summarizes the troubleshooting activities. Troubleshooting of the failed PCR reaction for Lassa virus [5]. As shown in Table 1 , all obvious possible measures were taken without success.
Even measures that were not the first choice failed to restore the PCR assay. Only when we used a basic one-step RT-PCR reaction kit from another manufacturer B, we could observe amplification as expected. At this point we need to point out that the Lassa virus PCR assay was well validated with the preferred basic PCR kit from manufacturer A which turned out to be superior to the kit from manufacturer B in terms of detection limit in our validation process.
Hence, this result was unexpected and surprising. However, it led us to the idea to test an aliquot from an old batch of the kit from manufacturer A and we were again surprised that this old aliquot worked as expected. While the old batch worked for every single one of our assays tested, the new batch only worked for some assays, e. Interestingly, considering the rules for PCR design and assessment, we were unable to identify any physical parameter of the primers like Tm, GC-content, Hairpin Tm or degree of degeneracy that would allow us to predict the sensibility of an assay to obviously slightly changing reaction conditions with the new batch.
However, not all of the 40 assays can be compared, for economic reasons and because of a restriction in batch size and stability. So far, we have been assuming that the proof of functionality of a new reagent batch for an assay selection is sufficient evidence of the quality of the new reagents batch.
This does not seem to be true for all individual assays. Therefore we wanted to share the lessons learnt with the PCR community, particularly with those who need to rely on rapid PCR-based diagnostic results, hoping to add a new troubleshooting criterion to the recent check list. According to the manufacturer of kit A, the new batch passed the QM check and no other customers reported similar problems.
However, fair enough, the company fully compensated the lost reagents. Summing up, we found that i different PCR assays show individual sensitivity toward changes in the reaction mixture. Again, we are not talking about new reaction conditions, but about expectedly the same reaction conditions and just a batch change of the same product.
If the extension time is too short, there will be insufficient time for complete replication of the target. If the annealing time is too short, primers do not have enough time to bind to the template.
If the annealing temperature is too high, primers are unable to bind to the template. If the denaturation temperature is too low, the DNA will not completely denature and amplification efficiency will be low.
If the denaturation time is too long, DNA might be degraded. If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will be low. GC-rich PCR products are difficult to amplify. To improve amplification, increase the annealing temperature. Template may be sheared or may contain PCR inhibitors.
If inhibitors are suspected, dilute existing template; otherwise, use fresh template and increase cycles. Try a control reaction in which you use a pure plasmid with the addition of the template to determine if any inhibitory effects exist. Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0. Insufficient amplification can result if the initial amount of template is too low.
Increase the number of amplification cycles in increments of 5, or, if possible, increase the amount of template. Use high-quality dNTPs. Using an excessive concentration of primers can increase the chance of primers binding nonspecifically to undesired sites on the template or to each other. Use well-designed primers at 0. In addition, verify that the correct concentration was supplied by the manufacturer.
If the primer concentration is too low, annealing may be inefficient. If the polymerase concentration is too low, not all PCR products will be fully replicated.
The optimal enzyme concentration depends on the length and difficulty of the template. Primers were designed or synthesized incorrectly by user or manufacturer. Verify that primers have the correct sequence and are complementary to the template. Use a primer design program to avoid repetitive sequences, regions with high complementarity, etc.
Perform a BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended regions. Use the tool at www. Use the lowest T m of the primers. Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water. Insufficient or omitted magnesium will result in no or reduced PCR product. Use 1. Use adequate units of enzyme. If you think the enzyme may be inactive, run a PCR with fresh polymerase from a different batch.
Causes Related to Cycling Times and Temperatures. Excessive cycling increases the opportunity for nonspecific amplification and errors.
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