Salam everyone,
So I am trying to extract DNA from isolates of clinical samples of staphylococcus aureus, I am using a QIAamp UCP kit. I followed the same manufacturer protocol and used Qubit to measure the concentration, what I am getting is 0.
I also tried to do some modifications by introducing extra heating steps, and bead beading, but I still don't get any?!
Has anyone had this issue before?
Healthcare-associated infections present an overall rate prevalence of 34.5% in patients in intensive care units in the USA1. This sector experiences a higher rate of healthcare-related infections. Most infections are associated with the use of invasive devices (catheters, bladder catheterization, mechanical ventilation, surgeries, and implants)2. Among the most common pathogens that cause these infections within intensive care units, according to the World Health Organization, is Staphylococcus aureus, representing more than 20% of isolates2.
In most cases, this pathogen is detected using microbiological cultures of samples taken from body fluids or therapeutic devices3. However, this method is not entirely satisfactory, as it is associated with ambiguous results3. This study compared five S. aureus deoxyribonucleic acid (DNA) extraction methods for detection by the polymerase chain reaction (PCR).
Strains of S. aureus ATCC 25923 were provided by Newprov Ltda (Paraná, Brazil). The strains were cultivated using Brain Heart Infusion medium and kept in a shaker incubator (New Brunswick Scientific, USA) overnight at 37°C. Culture aliquots of 1,000 µL were centrifuged at 10,000rpm for 3 min, and the pellets were frozen at -20°C.
The five conventional DNA extraction protocols4-7 compared here were selected because they are of low cost and easy to use. For each method tested, DNA was extracted in triplicate from independent samples of strands of the strain.
Protocol 1 used a bacterial lysis buffer (Tris-HCl) with Mg²+ and Ca²+ cations and glucose, and solution II, which contained a mixture of sodium hydroxide and sodium dodecyl sulfate (SDS), followed by purification with phenol:chloroform, as previously described4. However, the following adaptation was made for this work: proteinase K was not added, and the sample was freeze-thawed on dry ice and in a 70ºC water bath ten times, before purification with phenol and chloroform. The advantages of this method are that it is readily available and its ease of preparation4.
Protocol 2 used the GTSTM kit (Phoneutria) in accordance with the manufacturer’s instructions and the extraction was subdivided into 2a and 2b. In a, 20µL of Lifton buffer [0.2M sucrose, 0.05M ethylenediaminetetraacetic acid (EDTA), 0.1M Tris, and 0.5% SDS] and 1.2µL of proteinase K (10mg/ml) were added to the pellet. Then, the mixture was incubated at 60°C for at least 90 min (or overnight) to test the effect of the use of these reagents prior to using the kit. While in b, extraction was performed using the GTS kit. This method did not use phenol or chloroform.
Protocol 3 used cetyltrimethylammonium bromide (CTAB), as described in the literature5. It used proteinase K (20mg/ml), 10% CTAB, and ribonuclease (RNase) (10mg/ml) to enhance the DNA yield, followed by purification with phenol, chloroform, and isoamyl alcohol (25:24:1).
In protocol 4, the pellet was diluted using phosphate-buffered saline (PBS), pH 7.2 (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, and 1.4mM KH2PO4), followed by purification with phenol:chloroform (1:1)6.
In protocol 5, the pellet was diluted using NET buffer (50mM NaCl, 125mM EDTA, and 50mM Tris-HCl) and the addition of a denaturing solution, made up of 2.6N NaOH and 24% SDS, in accordance with a protocol described previously7.
The DNA concentration was measured using a Qubit® 2.0 Fluorometer (Life Technologies, USA). Using a spectrophotometer (Thermo Scientific NanoDropTM 2000/2000C, USA), the absorbance at 260nm and 280nm was used to assess the quality and purity of the extracted DNA. High values for both ratios (260/280> 1.8, 260/230> 2) are commonly accepted as a good indicator of pure DNA8. In contrast, a ratio of less than 1.8 is indicative of protein contamination, while a ratio of greater than 2.0 indicates contamination with ribonucleic acid (RNA)6. A low 260/230 ratio may indicate the presence of organic compounds, such as phenolates, thiocyanates, carbohydrates, or salts, in the extract8.
Qualitative polymerase chain reaction (PCR) was standardized using a total volume of 25µL, containing 8.5µL of ultrapure water, 12.5µL of pre-mix (buffer IC, Taq DNA, and deoxynucleotides - dNTPs), 1µL of each primer, and 2µL of DNA mold. The forward and reverse sequences of the 16S ribosomal RNA (16S rRNA) gene (5’-GACGGTCTTGCTGTCACTTA-3’ and 5’-AGTTCCAGTGTGGCCGATCA-3’, respectively) were used as primers to detect the amplified material (access GenBankPr016589760). The amplified product was approximately 119bp. The reactions were conducted in a thermocycler (Applied Biosystems Veriti® Thermal Cycler, USA), using the following procedure: initial denaturation for 5 min at 94°C, followed by 35 cycles (30 s at 94°C, 30 s at 52°C, and 30 s at 72°C), and final extension (10 min at 72°C). The amplified fragments were then analyzed by electrophoresis using a 1.5% agarose gel, stained with ethidium bromide (0.5µg/mL).
Tests were performed to determine the limit of detection of PCR using serial dilutions of genomic DNA, extracted from 103 colony-forming units per milliliter (CFU/ml) culture aliquots, plated on brain heart infusion (BHI) agar plates, to reproduce the minimum detectable level of CFUs9. In order to determine the sensitivity of the primer to lower DNA concentrations extracted from 103 CFU/ml, serial dilutions were prepared, and PCR was performed on the titrations using the same bacterial detection parameters.
The initial number of bacterial cells used for extraction with the 100µL culture solution was 4.7 x 107 CFUs for all of the protocols.
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