Comparison of four methods of extracting DNA from D. gallinae (: Dermanyssidae) Sophie Desloire, Claire Valiente Moro, Claude Chauve, Lionel Zenner

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Sophie Desloire, Claire Valiente Moro, Claude Chauve, Lionel Zenner. Comparison of four methods of extracting DNA from D. gallinae (Acari: Dermanyssidae). Veterinary Research, BioMed Central, 2006, 37 (5), pp.725-732. ￿10.1051/vetres:2006031￿. ￿hal-00903053￿

HAL Id: hal-00903053 https://hal.archives-ouvertes.fr/hal-00903053 Submitted on 1 Jan 2006

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Vet. Res. 37 (2006) 725–732 725 c INRA, EDP Sciences, 2006 DOI: 10.1051/vetres:2006031 Original article

Comparison of four methods of extracting DNA from D. gallinae (Acari: Dermanyssidae)

Sophie D, Claire V M, Claude C, Lionel Z*

Unité Mixte de Recherche PEV ENVL/INRA 958, Service de Parasitologie, École Nationale Vétérinaire de Lyon, 1 avenue Bourgelat, BP 83, 69280 Marcy l’Étoile, France

(Received 14 October 2005; accepted 31 March 2006)

Abstract – gallinae is one of the most serious ectoparasites of poultry and it has been implicated as a vector of several major pathogenic diseases. Molecular detection of such pathogens in is crucial and therefore, an important step is the extraction of their DNA from mites. So, we compared four DNA extraction protocols from engorged and unfed individual mites: a conventional method using a Cethyl Trimethyl Ammonium Bromide buffer (CTAB), a Chelex resin, a Qiamp DNA extraction kit and a more recent one filter-based technology (FTA). The DNA samples have been tested for their ability to be amplified by an amplification of a D. gallinae 16S rRNA gene region. The best results were obtained using CTAB and Qiagen methods at the same time with unfed and engorged mites (96% and 100% of amplified samples). FTA produced similar results when using unfed mites but not when processing engorged ones (96% and 70%). Finally, the Chelex method was the least efficient in terms of DNA amplification, especially when applied on engorged individuals (50%). The possible inhibitor role of these Chelex extracted DNA was demonstrated by the means of a PCR control on PUC plasmid. No difference was observed with CTAB, Qiamp DNA extraction kit or FTA methods using DNA extracted one year before.

D. gallinae / DNA extraction / engorged mites

1. INTRODUCTION to be an experimental vector of Coxiella burnetii, the bacteria responsible for Q The chicken or poultry red mite, fever [30]. Durden et al. [9] and Cham- (DeGeer, 1875) is a berlain and Sikes [6] have also proven cosmopolitan hematophagous obligate ec- that D. gallinae is able to transmit the toparasite of wild and domestic birds. It is eastern encephalitis virus by biting dur- the most economically important ectopar- ing blood feeding. This parasite has also asite of layer hens causing debilitation, been implicated in the vectorial transmis- decreased egg production and anaemia [7, sion of epidemic agents such as 15]. Furthermore, D. gallinae like other the fowlpox virus or the Newcastle disease have been involved as virus (NDV) where transovarian transmis- vectors of several major diseases (for sion was demonstrated [2, 23]. review [27]). Among human pathogenic To detect pathogens in vectors, molec- agents, D. gallinae has been demonstrated ular methods are useful tools that may be further developed. In Acari, these * Corresponding author: [email protected] techniques have been mainly applied on

Article available at http://www.edpsciences.org/vetres or http://dx.doi.org/10.1051/vetres:2006031 726 S. Desloire et al. species of economic and medical impor- performed on individual mites, frozen as tance such as , scabies mites and crop described. The concentration of each DNA pests [5, 13, 14, 16, 29]. Concerning the sample was determined by OD 260 and superfamily of Dermanyssoidea, the Lae- OD 280 measurements, with a biopho- lapidae and Varroidae families have been tometer (Eppendorf, Hamburg, Germany) the most studied from a molecular stand- and the purity was evaluated by the ratio point but these mites are not hematopha- OD260/OD280. geous whereas the presence of blood in D. gallinae could entail some additional diffi- culties [22]. 2.2.1. Cethyl Trimethyl Ammonium In addition to the detection of viruses or Bromide (CTAB) method bacteria in poultry red mites, these molec- ular methods would also lead to progress Individual mites were crushed with a in the knowledge of their genetics: popu- sterile plastic pestle in a 1.5 mL microcen- trifuge tube bottom containing 200 µLof lation genetics, gene transmission, pseudo ff arrhenotoky, mechanisms that are involved extraction bu er, (2% CTAB, 1.4 M NaCl, in the development of acaricide resistance 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, 0.2% 2-mercaptoethanol), preheated [3, 18]. All these studies require DNA and ◦ therefore the development of an efficient to 65 C. This homogenate was incubated at 65 ◦C for 1 h. Proteins were removed extraction protocol. In this study, we com- / pared four DNA extraction protocols based with one volume of chloroform isoamyl al- on different principles, from unfed and en- cohol. DNA was then precipitated from the gorged adult D. gallinae individuals. We aqueous layer by adding one volume of isopropanol, letting the sample stand for 2 tested the ability of these DNA extracts to ◦ be reliably amplified by a specific PCR am- or 3 h at –20 C and spun at 18 000 g for 30 min. The DNA pellet was then washed plification and then the same ability after µ one year of storage. with 300 L of ethanol 70%, dried for 10 to 15 min at 50 ◦C, and resuspended in 20 µL of ultrapure water. Tubes were placed for ◦ 2. MATERIALS AND METHODS 2hat4 C and DNA were resuspended again before being stored at –20 ◦C. 2.1. Mite samples 2.2.2. Chelex method Mites were collected from different poultry farms, in the Drôme, Ain, and A single D. gallinae mite was crushed Ardèche regions (Rhône-Alpes, France). using a P200 micropipette tip, in a mi- Individuals were kept in 70% ethanol and crotube containing 40 µLof5%Chelex ◦ frozen at −20 C, or directly frozen at resin (Instagene Matrix, Biorad, Hercules, ◦ −20 C. CA, USA). Suspensions were vigorously vortexed for 10 s, incubated at 56 ◦C for 35 min, vortexed again for 10 s, and 2.2. Mite DNA extraction methods incubated at 95 ◦C for 15 min. Suspen- sions were then vortexed and centrifuged For each DNA extraction method, 60 at 15 300 g for 3 min to allow easy re- mites (30 unfed and 30 engorged) were moval of the DNA solution from the top of tested, except in the case of DNA extracted the tube, with care taken to avoid remov- one year before where sample sizes ranged ing any Chelex resin from the bottom. This from 22 to 60 specimens. Extractions were DNA solution was collected in a 0.5 mL Method for DNA extraction from D. gallinae 727 tube, to be stored at –20 ◦C until PCR re- 2.3. Detection of DNA from D. gallinae action. by Polymerase Chain Reaction

The efficiency of DNA extraction meth- 2.2.3. Qiagen DNA extraction kit ods was evaluated by amplifying the mi- tochondrial 16S rRNA gene of D. galli- DNA extraction was performed using nae, using specific primers designed for the Qiamp DNA extraction kit (Qiagen, its 16S rDNA sequence (accession num- Hilden, Germany) according to the tis- ber L34326), F16 (5’TGGGTGCTAAGA- sue protocol, with some adjustments due GAATGGATG3’) and R16 (5’CCGGTCT- to the weight of one D. gallinae, about GAACTCAGATCAAG3’), which amplify 11 mg, compared to the recommended tis- a 377 bp region [4]. PCR reactions were sue weight of 25 mg: briefly, mites were performed in 25 µL volume containing crushed in 20 to 25 µLPBS,andvolume 0.8 µM of each oligonucleotide primer, was adjusted to 80 to 100 µL with ATL 100 µM of each dNTP (Amersham, Bio- buffer. Other reactive volumes were also Science, Buckinghamshire, UK), 2 mM of modified (10 µL proteinase K, 100 µLATL MgCl (Invitrogen, Carlsbad, CA, USA), buffer, 100 µL ethanol, 260 µLAW1and 2 2.5 µL10× Buffer (Invitrogen) and 1U AW2 buffer). The step with proteinase K of recombinant Taq polymerase (Invitro- was prolonged for at least 5 h, at 56 ◦C. Ge- gen), and 3 µL DNA extract, or the FTA netic material was finally eluted from the filter disk. PCR amplifications were per- column with 50 µLofAEbuffer. formed in a MWG thermal cycler (Biotech, Ebersberg, Germany), with a cycling pro- gram consisting of a 10 min denatura- 2.2.4. Filter-based technology (FTA) ◦ method tion step at 94 C followed by 35 cycles of denaturation (1 min, 94 ◦C), anneal- ◦ A single D. gallinae mite was crushed ing (45 s, 55 C) and extension (1 min ◦ against the microcentrifuge tube wall, us- 30 s, 72 C), and a final extension step of ◦ ing a P200 micropipette tip, and 20 µLPBS 10 min at 72 C. PCR products were sepa- pH 7.2 were added. This mix was homoge- rated by a 2% agarose gel electrophoresis nized by pipetting, and 5 µL was carefully containing ethidium bromide, and DNA applied on the FTA card, (Whatman, Mid- fragments were visualized under UV light. dlesex, UK) allowed to air dry for 2 h, and After a first amplification, each negative stored at room temperature. Disks measur- DNA sample was submitted to a second ing 1.2-mm were punched from the FTA one, and the results were scored. card (Harris, 1.2-mm micropunch, What- man) and placed in 1.5 mL microtubes. Disks were washed twice with 100 µLof 2.4. Test of an inhibitor role TE−1 (10 mM Tris; 0.1 mM EDTA) for 3 min, and air dried on a 56 ◦C heating A control PCR assay using M13 primers block for 10 min. Disks corresponding to to amplify DNA from PUC plasmid was engorged mites were first washed twice used to confirm the potential inhibitor role with FTA purification reagent (Whatman) of some mite DNA suspensions. PCR re- according to the manufacturer’s instruc- actions were performed in 25 µL, adding tions, before being rinsed with the TE−1. 100 fg of PUC DNA template, (quantity Disks were allowed to dry at 56 ◦Cfor previously determined as being close to the 10 min, and then used directly as templates detection limit), according to the protocol for a PCR amplification. described in a cloning kit (pCR II-TOPO 728 S. Desloire et al.

Table I. Number of amplified samples by PCR/total number of samples extracted from adult Der- manyssus gallinae by one method and submitted to the amplification (%).

CTAB method Qiamp kit Chelex resin FTA Unfed D. gallinae adults 30/30 (100) 30/30 (100) 25/30 (83) 29/30 (96) Engorged D. gallinae adults 28/30 (93) 30/30 (100) 15/30 (50) 21/30 (70) Total sample 58/60 (96) 60/60 (100) 40/60 (67) 50/60 (83)

TA Cloning kit, Invitrogen). Each of the mites, two methods, the CTAB and Qiamp negative samples in specific amplification DNA extraction kit, showed a better effi- of 16S rDNA of D. gallinae was added ciency with 96% and 100% of DNA sam- in the same proportion as in this D. gal- ples being amplified by PCR. These results linae specific amplification, i.e. 3 µL, to were significantly different from those ob- the PUC amplification mix. A positive con- tained with the FTA technique (83%) and trol reaction containing 3 µL of a positive Chelex resin (67%) (P < 0.05). If we con- DNA-Qiagen extract was performed. We sider only samples from unfed mites, the also tested the addition of 0.8 µg/µLof Chelex technique remained significantly Bovine Serum Albumin (BSA) to this lat- less efficient than the others (P < 0.05). est amplification mix, containing negative When comparing the four methods applied samples, in order to reduce the inhibitory to engorged mites, the CTAB and Qiamp effect of these samples. DNA extraction kit are still the most effi- cient methods. The engorgement was the cause of a decrease in efficiency of 33% 2.5. Statistical analysis and 26% respectively for the Chelex and Chi-square analysis was used to study FTA methods. In order to confirm the abil- the difference between methods and be- ity of such samples to inhibit amplification, tween engorgement status. P < 0.05 was 20 negative Chelex-DNA extracts for D. regarded as significant. gallinae amplification were added to the amplification mix with PUC DNA. Twelve modified the PUC amplification signal: 6 3. RESULTS lowered this signal and 6 totally switched off this signal. To remove the putative in- The concentrations of DNA extracted hibitory substances, 8 of the 12 negative with the Qiamp DNA extraction kit and Chelex suspensions were extracted a sec- CTAB were very similar with averages of ond time with the most successful DNA ex- 20.5 ng/µL and 43.5 ng/µL respectively. traction method, a Qiamp DNA extraction With the Chelex resin, the average con- kit, as described above. These re-extracted centration was 370 ng/µL, but the average samples were then added to the M13 PCR DO260/DO280 ratio of 1.3 indicated a control, and the detection signal of PUC high level of non-genetic materials in these DNA was restored for each amplification extracts. It was confirmed by the fact that reaction. The addition of BSA to five neg- no DNA signal was observed on an agarose ative Chelex extracts has also restored the gel. PUC DNA amplification (Fig. 1). We compared the efficiency of the four Compared to freshly extracted DNA extraction methods, defined as being their samples, DNA which have been ex- ability to produce DNA of a sufficient qual- tracted one year before did not show ity to be amplified by a PCR (Tab. I). With- any significant difference in their ability out considering the engorgement status of to be amplified (100% for Qiamp DNA Method for DNA extraction from D. gallinae 729

Figure 1. Test of the inhibitor role of negative DNA-Chelex extracts on M13 amplification of PUC. (A) Modification of PUC amplification by addition of some negative DNA-Chelex extracts. Lanes 1 to 7: the 103-bp M13 region of PUC was amplified from 100 fg of plasmid DNA, in the presence of 7 different negative DNA-Chelex extracts for D. gallinae specific amplification. Lane 8: the same PCR product, with positive DNA-Qiagen extract for D. gallinae specific amplification. T+: positive reaction control containing 100 fg of PUC DNA template. T- : negative control, with water as the template. L: 100 bp ladder (Invitrogen). (B) Effect of a Qiagen extraction on DNA-Chelex extracts which inhibited the PUC amplification (samples 1, 2, 4, 5, 6 from A). PCR product of the M13 region of PUC, when these Qiagen extractions were added to the mix. (C) Effect of BSA on the inhibitory effect of DNA-Chelex extracts on the PUC amplification (samples 6, 1, 2 from A). BSA was added at 0.8 µg/µL to the PUC amplification mix, in addition to negative DNA-Chelex extracts. extraction kit, 100% for CTAB and 93% using separation with a column [8,11], and for FTA). a Chelex resin [10, 14, 21, 25, 28, 29]. An- other attractive method, the FTA technol- ogy, was also tested, and compared to the 4. DISCUSSION others. It has been reported for detection of pathogenic DNA [19] and particularly for According to extraction methods al- the detection of microsporidia in fire ants ready published for various acari (Erio- and of Francisella tularensis in pools of phyidae, , Laelapidae, Phytosei- ticks, with satisfying results [12, 24]. idae, Tetranychidae, Trombidiidae), we The CTAB and Qiamp DNA extraction chose and compared three conventional kit protocols gave the best results in our methods for extracting DNA from D. gal- studies with an efficiency close to 100%, linae: a CTAB method, with a lysis step regardless of the engorgement status of followed by a phenol chloroform precipita- the mites, while with the Chelex method, tion [17, 20], a Qiamp DNA extraction kit only 67% of efficiency in amplification 730 S. Desloire et al. was obtained. These results were corre- protocol recommended for samples con- lated with the concentration and the pu- taining blood was used. This technique rity for each method: Chelex samples could be improved when applying it to seemed to be rich in DNA, but their blood-fed mites. For example, an addi- OD260nm/OD280nm ratio indicated an tional purification step prior to application important contamination by non genetic of the mite suspension onto the FTA card material, whereas concentrations of CTAB may be required, e.g. centrifugation to re- and Qiamp DNA extraction kit samples move larger debris, in order to overcome were lower, but with a ratio showing diminished sensitivity. that DNA was pure. When applied to One essential point in studies con- engorged individuals, the Chelex method cerning is the research of gave DNA successfully amplified in only pathogenic agents to assess their potential 50% of the cases. This suggests that vectorial role. Detection of pathogens in- the presence of blood, combined with a side mites may be carried out on blood-fed very simple extraction procedure, may in- specimens, artificially engorged or taken terfere with PCR amplification and this from breeding facilities. The Qiamp DNA possible inhibition was assessed in two extraction kit and CTAB methods are ways. First, amplification of PUC plas- shown by the present study to be the most mid DNA with M13 primers was regu- adapted but are time consuming. FTA tech- larly inhibited when some negative Chelex nology gives less satisfying results than do samples were added to this control PCR conventional methods (CTAB and Qiamp mix. In addition, the inhibitory effect of DNA extraction kit), but is rapid to per- these samples was overcome, by extract- form, less expensive and does not require ing them a second time with the Qiamp laboratory equipment, at least for collect- DNA extraction kit, or by using BSA, ing DNA samples. In spite of the loss of known for reducing the inhibitory effect 15% in efficiency, the FTA method could of haemoglobin [1]. However, the authors be interesting and would be the most suit- of studies using this Chelex resin did not able when it is necessary to analyse a large notice any difficulties but they worked on number of samples taken from breeding fa- non hematophagous mites (Tetranychidae cilities, since it permits the collection of and Phytoseiidae) [14, 21, 25, 29]. Some DNA samples in situ and facilitates their authors have already noted that DNA tem- storage and shipment prior to laboratory plate extracted by Chelex may not be suffi- analysis. Finally, we would not recom- ciently purified for standard PCR amplifi- mend the Chelex method in order to detect cation [13]. Moreover, the blood presence pathogen DNA in mites, since our experi- in our specimens, known to be a PCR ments demonstrated that samples extracted inhibitor, may have increased this prob- with this method contain PCR inhibitors, lem [26]. Schwartz et al. [22] demonstrated which could yield an underestimate of the that inhibitors of PCR amplification were presence of DNA pathogens. present in engorged ticks and they recom- We also observed that each of these mend the use of a sufficiently rigorous ex- methods is able to preserve DNA samples traction step to remove any potential PCR at room temperature for FTA and at –20 ◦C inhibitors. for the three others. During this study, we The FTA technology applied to unfed also noticed with the CTAB method, an mites obtained results similar to those with improvement in amplification results when CTAB and the Qiamp DNA extraction mite samples were preserved in alcohol kit. Decreasing efficiency was noted when and stored at –20 ◦C, prior to DNA ex- working on blood-fed specimens, while the traction, instead of being directly stored Method for DNA extraction from D. gallinae 731

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