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How do I avoid false positive and false negative

The false negative results usually come from bad DNA quality or failed PCR. Internal control is the best solution to prevent this problem. In IQ2000^TM system, internal control (house-keeping gene) is included and users can use the internal control to monitor the DNA quality and PCR procedure easily.

The false positive results usually come from contamination of the final PCR products or cross contamination during extraction and reagent-adding step. To avoid contamination from the PCR products, the best way is to isolate the gel electrophoresis room far away from the sample preparation and reagent preparation room. About the cross contamination problem, a good training is the best way to prevent it.

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How do I know the sensitivity is appropriate? Can I adjust the sensitivity?

You can monitor system's sensitivity by the quantified positive standard included in the kit. In IQ2000^TM WSSV system, you should dilute the standard to 10 copies/ul by yeast tRNA. Then you can monitor the sensitivity by the 10-copy standard. If the 10-copy standard resulted 3 bands (910, 550, and 296 bp), your system is over sensitivity. 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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Must I see the hose-keeping gene's product from the negative samples?

It depends on what kind of sample you want to test. Because the house-keeping gene was designed according to the shrimp genomic DNA, only the shrimp samples will show the right products. The negative shrimp samples must show the house-keeping gene; otherwise, you have to check the DNA quality to avoid false negative results. For other crustaceous animals, they might show the house keeping gene but the product size could be different from the 848 bp in some species. If the samples are pond dirt, pond water, or feed, you will not get the house-keeping gene.

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