Study On the Correlation Between Microbial Metazoan (animal) Compositions In Polymerase Chain Reaction (PCR) and Animal Number/Biomass.

The factors affecting PCR amplification (presence of soil, ethanol; annealing temperature; primer sequence) microbial metazoan (animal) as well as the correlation of PCR product composition to either the relative biomass of each animal species or the number of individuals of each species present in the original samples are studied under simulated laboratory experimental conditions.  Three different samples: animal only (ants, annelids, nematodes, rotifers and tardigrades); mixture of animal and soil; mixture of animal, soil and ethanol were used.  Terminal Restriction Fragment Length Polymorphism (T-RFLP), Length Heterogeneity PCR (LH-PCR) and sequencing were applied to detect animal DNA.  Two forward primers 18S11C and 18S11M combined with two reverse primers 18S2a and 18S0R were used to compare the primer bias in PCR amplification of animals.  Annealing temperatures of 55° C and 50° C were introduced to detect the stringency of primers in PCR amplification.  Results show that the percent of amplified PCR product performs higher correlation with the number of animals than with the number of the biomass.  DNA samples of each animal were mixed in pair to detect if any particular animal DNA was inhibited in PCR by others or not.  In the combination of nematode and ant, nematode was both amplified at 55° C and 50° C using 18S11M-18S2a but not by 18S11C-18S2a.  In the combination of nematode and annelid, nematode was amplified at 50° C but not at 55° C.  The results suggest that both primers and temperatures affect the composition of amplified PCR products and provide valuable information for future small soil animal composition studies.