Identification of forensically important blowfly species (Diptera: Calliphoridae) by high-resolution melting PCR analysis

Abstract

We describe here the successful coupling of real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis to rapidly identify 15 forensically important species of blowfly from the family Calliphoridae (Diptera), which occur in Poland. Two short regions (119 and 70 base pairs, respectively) of cytochrome oxidase gene subunit I with sufficient sequence diversity were selected. In the case of lacking taxa (e.g., reference species) these amplicons can be synthesized using sequences deposited in gene banks. The technique utilizes low template DNA concentration and is highly reproducible. The melting profile was not altered up to a 10,000-fold difference in DNA template concentration (ranging from 5 pg to 50 ng). The several HRM runs performed on different specimens from Poland belonging to the same species and on different days resulted in only minor variations in the amplification curves and in melting temperatures of the peaks. Intraspecific variation in a larger scale was tested using synthesized oligonucleotides from cosmopolitan Lucilia illustris originating from Poland, France, Great Britain, India, and USA. As HRM PCR analysis is sensitive to even single base changes, all geographic variants of this species were identified. This technique is also cost-effective and simple, and it may even be used by non-geneticists. A working protocol was ultimately constructed for the purpose of rapid and accurate species identification in most countries in Europe regardless of which stage or which part of a blowfly was collected.

Factors Affecting Species Identifications of Blow Fly Pupae Based upon Chemical Profiles and Multivariate Statistics

“…Any damaged or dead pupae will never be identified by this method, and few crime laboratories have the resources available to rear flies. An alternative is to extract the DNA from the pupae and determine the species typically by sequencing a mitochondrial DNA locus [11,12,13,14,15,16,17], however, expertise is necessary for the analysis of the DNA sequence, and a thorough representation of all possible species is required [18]. …”

Rapid discrimination between four Antarctic fish species, genus Macrourus, using HRM analysis

“…Some previous studies have found this technique sensitive to the quality and quantity of DNA used in the QPCR (Haynes et al, 2009) while others, as with this study, found the template DNA had little effect on the success (Malewski et al, 2010). Dilution of the DNA to a standard concentration was not essential to produce successful results however it did improve the quality of the data.…”

“…This technique has already been used to screen for genetic variation in insects, mammals, human disease, and fish (e.g. Berry and Sarre, 2007;Dobrowolski et al, 2009;Haynes et al, 2009;Malewski et al, 2010;McGlauflin et al, 2010;Vossen et al, 2009) and found to be fast, low cost in terms of consumables and reliable. Also, unlike conventional genetic screening by sequencing, it could be carried out at field stations or onboard ships.…”

Maggots reveal a case of antemortal insect infestation

“…Another application is the detection of toxic substances in insects feeding from dead persons' tissues [5]. There have been several studies done according to the biology of these animals' living associated with corpses as well as their application in legal investigations in the last years [6][7][8][9][10][11][12].…”