Defining specificity in DNA detection of wildlife: Response to Gonçalves et al. "the risks of using "species-specific" PCR assays in wildlife research: the case of red fox (Vulpes vulpes) identification in Tasmania".
نویسندگان
چکیده
Gonçalves et al. [1] report an examination of the PCR-based species identification test used to detect foxes from faecal material in the ongoing fox eradication programme in Tasmania. The authors rightly point out that poor design in screening methods may lead to false negative or false positive results and they sought to ‘‘better evaluate the specificity of the putatively fox-specific pair of PCR primers’’ as reported in Berry et al. [2] and applied by Sarre et al. [3]. We welcome the scrutiny of the test as DNA-based tests are being used more frequently in wildlife detection [4] and yet estimation of test specificity and sensitivity is rarely attempted. Unfortunately, Gonçalves et al. [1] have undertaken an analysis of an incomplete imitation of the test we applied. In doing so, they reach conclusions that do not apply to the test as it is conducted in practice nor do they actually estimate test specificity. Taken at face value, the findings by Gonçalves et al. [1] could lead to erroneous interpretations, as was the case in a recent publication in which their findings were cited as support for claims of a high rate of false positives in the detection of Tasmanian foxes [5]. The test for fox DNA [2,3] uses a sequential two phase approach aimed at determining, with acceptable probability (Ramsey et al. in review), whether a scat contains fox DNA or not. The first phase of the test comprises an initial screening of DNA through amplification with the primers VV-cytb F and VV-cytb R [2]. These primers preferentially amplify fox mitochondrial cytochrome b DNA and are used to identify samples that are likely to contain fox DNA on the basis of the size of the fragment amplified. They are used as part of a multiplex PCR incorporating a second ‘universal’ primer pair targeting mammalian mitochondrial 12s ribosomal DNA as a positive test for PCR amplification. If the ‘universal’ primers amplify a product but the primers targeting fox cytochrome b DNA do not, then the lack of amplification of possible fox DNA cannot be attributed to PCR failure and is most parsimoniously interpreted as meaning that fox DNA has not been detected. The second phase of the fox DNA detection test is conducted only if a PCR product of the size of the target cytochrome b fragment is detected in the multiplex reaction and involves direct sequencing of the PCR product obtained from a second amplification using the VV-cytb F and VV-cytb R primers only. A scat is considered to contain fox DNA only when a direct match is found between the sequence obtained from the fragment amplified and published fox sequences [2,3,6]. This sequential two phase approach, which is described explicitly in Sarre et al. [6], was adopted to make cost effective the screening of predator scats in the large numbers typically required for the
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ورودعنوان ژورنال:
- Forensic science international. Genetics
دوره 13 شماره
صفحات -
تاریخ انتشار 2014