Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections

Abstract

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes.

Figure 1

Neosomes of some tungids. A) Tunga travassosi, female. Bar: 8 mm B) Tunga caecata, female. Bar: 12 mm.

Figure 2

Amplification, without the use of the MDA technique, of the 200 bp fragment of the 16S mtDNA of samples from scientific collections preserved in 70% ethanol and stored at room temperature. 1) 100 bp DNA Marker; 2) Tunga penetrans (control); 3) Ctenocephalides felis felis (control); 4) C. felis felis (2008); 5) T. penetrans (2007); 6 and 7) T. caecata (2005 and 2007); 8) Negative control.

Figure 3

Amplification, with the use of the MDA technique, of the 200 bp fragment of the 16S mtDNA gene of samples from scientific collections preserved in 70% ethanol and stored at room temperature. 1) 100 bp DNA Marker; 2) Tunga penetrans (control); 3) Ctenocephalides felis felis (Control); 4 and 5) T. penetrans (2007 and 2002); 6 and 7) T. travassosi (1966 and 1916); 8, 9 and 10) T. caecata (2007, 1980 and 1989); 11 and 12) T. terasma (1996 and 2003); 13) T. bondari (1909); 14 and 15) Tunga sp. (2000); 16) C. felis felis (1988); 17) Negative control.

Figure 4

Analysis of sequence quality of the 16S mtDNA gene fragment using the CodonCode Aligner software. The black line denotes Phred = 20. A) Tunga penetrans; B) Ctenocephalides felis felis; C) T. caecata; D) T. travassosi; E) Tunga sp.; F) T. terasma.

Figure 5

Analysis of similarity between some of the sequences (177 pb) of the 16S mtDNA gene fragment from fleas preserved in scientific collections and sequences deposited in GenBank, as analyzed using the BLASTn tool. A) Tunga penetrans collected in 2002; B) T. bondari collected in 1909.