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 What dose" PCR negative results" mean?
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First, we should define the "detection
limit". The "detection limit" in a PCR system means a
specific viral template amount which can be detected repeatedly by this PCR
system when this amount or higher is presented in the sample. For IQ2000TM
WSSV system, the detection limit is 10 viral copies per reaction.
When the viral particle amount is higher than the
detection limit, we will be able to get a PCR positive result. If there is
not any virus particle inside the reaction tube, the result must be negative. But
when the viral amount is lower than the detection limit, such as 3 viral
particle in a reaction tube, the result will become a statistic issue; the
result will be sometimes positive and sometimes negative.
So, the PCR negative means the specific sample is virus
free or its viral amount is lower than the detection limit.
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How do I know my PCR cycle is operating under proper condition?
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First,
please ask the machine supplier to tune up your cycler every
year. Then, you can monitor the PCR efficiency from the 10-copy
standard (WSSV system). If the 10-copy standard resulted 3 bands (910,
550, and 296 bp), which means the cycler is over sensitive. You may reduce
2 to 3 cycles from the first reaction. On the other hand, if the 10-copy
standard did not result any band, you may increase 2 to 3 cycles on the first
reaction to increase the sensitivity.
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What's the advantage of the nested PCR?
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There
are two major advantages of the nested PCR design. First, it can increase the
sensitivity. For the viral diagnostic purpose, sensitivity is a very
important requirement. Second, nested PCR can increase the specificity;
only the right products from the first reaction can be amplified in the nested
reaction.
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