When we are trying to estimate if there is any positive animal within a big unknown population, we need to sample and check 150 animals individually to reach 95% confidence level.   But it is not practical to screen individually, also the capacity of each PCR reaction is limited.  The following descriptions could explain why.

Optimized DNA concentration for IQ2000^TM system:  200ng/reaction.

200ng almost = 40,000 cells, in other words, we can check 40,000 cells / PCR reaction.

Detection limit of IQ2000^TM system: 10 viral copies/reaction.  Thus, if there is one virus within 4000 shrimp cells (10 in 40000), we will be able to detect it reproducibly.

Latent phase: the host has been infected, but the viruses haven't started reproducing themselves and thus, they wouldn't transmit the disease to other hosts.

Reproductive cycle: the viruses have started reproducing themselves.  Finally, the host cell lysed and the viral particles released. The released viruses could infect other cells and also other hosts.  Stress can trigger off the viruses from the latent phase to the reproductive cycle.  

How can we know the situation is upon the latent phase or in the reproductive cycle?  The infectious level will be the key to this question.  In reproductive cycle, the viruses have started to reproduce themselves and we can find more viruses within that sample (medium or severe infected by IQ2000^TM system). From the experimental data, in the WSSV case, when the virus/cell ratio is above 1 in 400, the viruses might be moving to the reproductive cycle.

Now, let's go back to the PL case.  If we detect those 150 animals together and there is a medium infected PL with a 1 (virus) in 400 (cells) case within 149 negative PLs. That infected PL has already moved to the reproductive cycle and it could infect the other PLs in the future.  When we grind all of them together, the virus/cell ratio will drop from 1/400 to 1/60000.  Recall that the capacity of PCR is only 10 viruses in 40000 cells (1/4000).  Thus there is a big chance that we will get a negative result in this case.

Let's check another case.  If we only check 30 animals, the virus/cell ratio will drop from 1/400 to 1/12000. It is still lower than 1/4000, unless the system's sensitivity (or detection limit) can be improved to 3 copies/reaction (or 3 in 40000). Fortunately, the IQ2000^TM system can detect 3 copies in most cases even though we just claimed our detection limit is 10 copies.

Due to we still have to fulfill the statistical fact (150 animals) and PCR capacity (30 animal maximum) both, we recommend separating those 150 animals into 5 groups and detect them separately.  If there is any positive result within those 5 groups, there is infected animal within the whole population.

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