Evaluation of three methods for the extraction of dna of Salmonella sp from artificially-contaminated chicken eggs

Introduction

Faced to the problem of the presence of Salmonella sp. in eggs and its importance in public health, it is necessary to implement effective control measures. Aimed at ensuring the safety of foodstuffs for human consumption (Berchiere, 2000). In this context, we stress the importance of seeking new technologies that bear rapid results with high sensitivity and at low cost for the detection of this bacterium.

The polymerase - PCR chain reaction appears to be a useful strategy for the detection of bacteria of the genus Salmonella in different substrates such as meat, blood, milk and eggs. For the use of the polymerase chain reaction technique, it is essential to obtain good quality DNA in order to achieve good tests results, in which the excess of cellular structures and proteins can inhibit the amplification process. DNA extractions should provide good band standardization, with sufficient quantity and quality so as not to cause interference in the electrophoresis gels migration patterns.

That means that new protocols still need to be tested for different substrates, with the aim of developing new technologies and even improve the existing ones. For this reason, the objective of this study was to assess the effectiveness of three methods for the extraction of Salmonella sp. DNA from Chicken eggs free of specific pathogens (SPF).

Materials & Methods

Salmonella sp and Escherichia coli samples were lyophilized at the Avian Health Research Laboratory, Federal University of Viçosa. These bacteria were activated in brain- and heart infusion broth (BHI) at 37 ° C for 18 hours.

75 white, free of specific pathogens (SPF) chicken eggs were used, they were divided into 5 groups of 15 eggs each. The eggs were inoculated in the allantoidal cavity: Firstly, each egg was disinfected with iodine solution tincture 10% at the drilling site of shell, then the eggs were drilled in the air chamber with the help of a stainless steel needle adapted to a 30 x 12 mm gauge rubber piece The eggs were inoculated with a 1 mL, 0.1 mL tuberculin syringe. The eggs were inoculated with bacterial cultures, three groups of these were inoculated with Salmonella sp. at a concentration of 10⁵ CFU/ml, another group was inoculated with a strain of E. coli in the same concentration as above and the eggs in the control group were subject to the same procedures using 0.85%, buffered and sterilized saline. For the inoculation, a needle of 12.7 X 0.33 mm caliber was used; it was introduced in the air chamber, placing the content in the alantoid cavity, approximately at an angle of 30 °. After inoculation, the holes were sealed with glue and the eggs were transferred to trays, where they stayed for 24 hours at room temperature.

After this time, the yolks of each group were collected aseptically and placed in a sterile beaker and homogenized. Then, 10 ml of the homogenized yolk were transferred to the centrifuge tubes.. They were centrifuged at 2.500 x g at room temperature. The supernatant was discarded, and the sediments were reintegrated in the suspension at equal volume with PBS pH 7.2. and once again centrifuged, and the supernatant was, again, removed. Sediment obtained from each group was stored at - 20 ° C until DNA extraction.

The extraction of bacterial DNA using the commercial kit - GenElute Bacterial Genomic DNA (Zigma^®) was performed according to the manufacturer''''s instructions. The obtained DNA was quantified and analyzed in agars gel (1%); it was stained with ethidium bromide and viewed under ultraviolet light.

The other two protocols using silica particles with DNAzol or NaI were based on the studies by Boom et al. (1990).

600 µL of NaI (or Dnazol), were added to the samples; they heated to 55 ° C and stirred gently for five minutes. The material obtained was centrifuged for 5 minutes at 12.000 x g at room temperature and the supernatant was collected using a pipette. Then, 50 μl of silica suspension were added to the mix (silica dioxide-Sigma Chemical Co., St. Louis, Mo) ant the mixture was homogenized with a vortex. The mixture was incubated in an end-over-end shaker (Speci-Mix, Thermolyne) for 10 minutes at room temperature, then it was centrifuged for 30 seconds at 12.000 x g at room temperature, the supernatant was discarded by tube inversion. The sediment was rinsed twice using 1 mL washing buffer solution (50%, 50 mM Tris-HCl pH 8, 10 mM EDTA pH 8). After centrifugation for 30 seconds at 14.000 x g at room temperature, all the washing buffer solution was removed using a pipette.

1 mL of acetone was then added and homogenized in the vortex and centrifuged for 30 seconds at 14.000 x g at room temperature. The supernatant was discarded and the residue of acetone was evaporated from the sediment through the open-lid tube at a temperature of 56 ° C for 10 minutes. DNA adhered to the silica was eluted by adding 50 μl of TE (5 mM TRIS-HCl pH 8, 0.5 mM EDTA pH 8), incubated at 50 ° C for 5 minutes and centrifuged for 30 minutes at 14.000 x g at room temperature to solidify the sediment. The supernatant was discarded using a pipette, the DNA samples were extracted, analyzed and quantified by a NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington DE, USA) spectrophotometer reading, which estimates the amount of DNA in the sample in μg / μl and the quantity of the material by the value obtained in the ratio of DO260nm / DO280nM.

With the aliquots of each DNA sample, obtained from the two extraction protocols, the amplification reactions were performed, with a final volume of 50 µL containing: 2µL of DNA, 5 µl of buffer 10 X (200 mM Tris-HCl, pH 8, 500 mM KCL - Invitrogen^®) 1.0 µL of DNTP at 10 mm (Datp, dTTP, Dctp, Dgtp - Invitrogem), 1.5 µl of MgCl2 at 50 mM, 1µL of each primer at 25 pmol, 0. 3µL Taq polymerase at 1U/µL and ultra pure water q.s.p.

The pair of primers for DNA amplification was synthesized based on the sequence: 5'''' GTA AAA TTA TCG CCA CGT TCG GGC AA 3'''' (primer 1) e 5'''' TCA TCG CAC CGT CAA AGG AAC C 3'''' (primer 2), to amplify a fragment of 284 pairs of invA gene base of Salmonella sp. The PCR reaction was performed on all extraction samples in a thermal cycler. Amplification conditions went from an initial cycle of denaturalization at 94° C for 5 minutes, followed by 30 cycles of denaturalization at 94° C for 30 seconds, heated to 60° C for 30 seconds and then the temperature was increased to 72° C for 30 seconds and, finally, the temperature was increased to 72° C for 7 minutes. The detection of the PCR products was made by electrophoresis in agars gel at 1%. As a molecular weight marker a 100pb DNA ladder (GibcoBRL) was used. The display was made by means of a UV transluminator.

Results and Discussion

Although extraction of bacterial DNA was possible using the protocols in which silica particles are used, together with the DNAzol or NaI reagent, these reactions were not the desired ones. Samples showed values lower than 1.8, indicating protein contamination of the DNA. Therefore, it is necessary to make adjustments in the extraction protocol.

As for the commercial kit - GenElute Bacterial Genomic DNA kit (Zigma^®), it was not possible to visualize the amplification of DNA in the extracted samples. However, it was possible to view the amplification in all the samples obtained through the PCR protocols. The primers derived from the invA gene were used, obtaining a band of DNA amplification with 284 pairs, as expected in all samples of Salmonella sp., still without observing any amplification of E. coli. DNA Based on this, we can state that the invASalmonella sp. gene contains unique sequences and showed that this gene is a suitable target for PCR, with possible applications in Salmonella sp. diagnosis.

The best result was obtained when the DNA was extracted using silica particles with NaI, due to the presence of more intense DNA bands, indicating greater representation of genetic material (Fig. 1). Therefore, DNA extraction by this method can be used safely in PCR testing, thereby reducing the costs of the technique, mainly in terms of the purchase of DNA extraction kits.

Rahn et al., (1992), using a pair of oligonucleotides of the invA gene, were able to detect 626 strains of Salmonella in 630 tests from a colony isolated and incorporated directly into the PCR. They achieved a 99.4% sensitivity and specificity of 100%, since they noted no positive reaction in any negative control belonging to other 33 evaluated enterobacteria.

According to Rohland & Hofreiter (2007), the extraction method using silica particles has some advantages over other protocols, since it is quick and easy, and small amounts of the sample may be used. Furthermore, it is very easy to implement, since it uses standard laboratory equipment and few chemicals. On the other hand, it offers efficient removal of PCR inhibitors.

Figure 1. Electrophoretic pattern of products amplified by PCR of Salmonella sp DNA, extracted by methods using silica particles with DNAzol^® and NaI

M: Molecular weight marker of 100pB; EC. E. coli sample; SO,S1,S2,S3 Salmonella sp samples; C+: Positive control of a Salmonella sp culture; DNAzol DD: Negative control; B: White.

Conclusion

The Protocol that uses the silica particles permits the extraction and mining of genomic DNA of Salmonella sp of good quality, integrity and suitable for amplification.

Bibliography

Berchiere Junior A. 2000. Salmoneloses aviárias. pp. 185-195. In: Doenças das Aves. Berchiere Junior A, Macari M (ed.). FACTA, Campinas, Brasil.

Boom R, Sol CJA, Salimans MMM, Jansen PM, Wertheim PME, Noordaa JVD. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28: 495-503.

Rohland N & Hofreiter M. 2007. Ancient DNA extraction from bones and teeth. Nature Protocols 2:1756-1762.

Rhan k, De Grandis SA, Clarke RC, McEwe SA, Galán JE, Ginocchio C, Curtis R, Gyles CL. 1992. Amplification of invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol. Cel. Probes 6:271-279.