An alternative DNA extraction method for detection of Blastocystis spp. in human fecal samples.

Polymerase chain reaction (PCR) is an effective technique for diagnosis of Blastocystis infection. Notably, DNA isolation procedure is extremely critical for the PCR step. In the present study, a recently described extraction procedure, named as the "sand method" was modified and adapted for isolation of Blastocystis DNA. To evaluate its efficacy, the current method and QIAamp DNA Stool Mini Kit (Qiagen) were applied to fresh human stool samples. Our results indicated that, the mean DNA concentrations obtained by the sand method and the commercial kit were 48 and 55 ng/µl, respectively. Also, no DNA inhibitors were detected in two methods. The sand method was capable of detecting 16 parasites per 50 mg feces. DNA samples extracted by both methods were subjected to PCR. Blastocystis spp. were detected in 11 (31.4%) of 35 samples, and perfect agreement (κ: 1.000) was found between the PCR-sand method and PCR-commercial kit method. The samples that were detected positive by PCR-sand method were successfully sequenced, and Blastocystis subtypes (STs) were identified as ST3, ST2 and ST1. In conclusion, the present study indicates that the sand method provides a simple, rapid and inexpensive procedure for reliable extraction of Blastocystis DNA from stool samples.

[A new method for the disruption of cell walls of gram-positive bacteria and mycobacteria on the point of nucleic acid extraction: sand method]. / Nükleik asit ekstraksiyonunda kullanilmak üzere, gram-pozitif bakteriler ve mikobakterilerin hücre duvarlarinin ortadan kaldirilmasinda yeni bir yöntem: Kum yöntemi.

Nowadays molecular methods are widely used in the rapid diagnosis of infectious agents. Polymerase chain reaction (PCR) is the most preferred method for this purpose. Obtaining sufficient and pure DNA or RNA is important for the PCR. Different DNA extraction protocols such as phenol-chloroform, proteinase K, glass beads and boiling have been used successfully for DNA isolation from gram-negative bacteria. However since gram-positive bacteria have a thicker layer of peptidoglycan and mycobacteria have complex glycolipids in their cell walls, for the isolation of DNA or RNA from these microorganisms, the complex cell wall structure must be eliminated. For this purpose, the bacterial cell wall must be completely or partially removed forming sferoblast using lysostaphin in the Staphylococcus genus as gram-positive bacteria and using a chemical like cetyltrimethyl ammonium bromide for the Mycobacterium genus. In this study, we planned to use sand particles for the mechanical elimination of the cell wall without any need for chemicals and we called this procedure as "sand method". For the purpose of DNA extraction, the fine-grained sand was washed with ddH(2)O without losing small particles and then sterilized by autoclaving. For the purpose of RNA extraction; the sand was washed with ddH(2)O, incubated for 30 minutes with 10% HCl, and then autoclaved. A methicillin-resistant Staphylococcus aureus (MRSA) strain previously isolated and identified from a clinical specimen was mixed in 100 µl Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. The MRSA-sand mix was treated with proteinase K and phenol-chloroform, and ethanol precipitation protocol was then followed for obtaining DNA. For comparison of the sand method with the other methods, the same amount of bacteria used in the sand method was incubated for one hour with lysostaphin, and then the proteinase K DNA extraction method were completed in the same way used in the sand method. For obtaining RNA from M.tuberculosis H37Rv ATCC 25618, M.tuberculosis H37Ra ATCC 25177 and M.tuberculosis H37Rv Pasteur Institute RSKK 598 standard strains, bacteria were dissolved in 20 µl Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. After that, the classic RNA extraction protocol using guanidinium thiocyanate-phenol-chloroform (GTPC) was completed. To investigate the usefulness of the obtained DNA, a PCR was performed with specific primers for staphylokinase and enterotoxin genes that were shown in the genome of the chosen MRSA strains from our previous studies. To investigate the usefulness of the obtained RNA from the sand method; first cDNA synthesis is completed. The PCR efficiency was then tested using primers specific to the efflux pump genes of M.tuberculosis including Rv1410c, Rv2333c, and DrrA genes. To compare the effect of the sand method, GTPC protocol was applied in the same amount of mycobacteria without the sand treatment. The DNA obtained from MRSA with the application of lysostaphin and the DNA obtained from MRSA by the sand method were run in agarose gel electrophoresis. The amount and purity of DNAs were measured with a spectrophotometer. The same amount and purity of the DNAs were approximately the same in both of the extraction methods. The existence of non-inhibitors of DNA in the sand method was shown with the PCR, which have worked efficiently with the DNAs obtained from the sand method. RNA was obtained efficiently from the Mycobacterium strains by the sand method, but no RNA could be obtained from the mycobacteria with the other methods. It was shown that the RNA obtained using the sand method worked effectively in both cDNA synthesis and PCR in which synthesized cDNA was used. The sand method described in the study worked effectively to obtain sufficient amount of pure DNA and RNA from the bacteria containing rigid cell walls that are difficult to obtain the nucleotide. It was concluded that, using the sand method instead of relatively expensive lysostaphin or other chemicals, has important advantages such as decreasing the cost and the shortening of the DNA extraction period.

Blastocystosis in post-traumatic splenectomized patients.

BACKGROUND/AIMS: The aim of this study was to determine the prevalence and significance of intestinal protozoa, specially Blastocystis spp., and to perform PCR-based subtype classification for understanding the importance of Blastocystis spp. in the pathogenesis of gastrointestinal disorders in post-traumatic splenectomized patients. MATERIALS AND METHODS: A total of 60 stool samples were obtained from 30 post-traumatic splenectomized patients and 30 healthy controls. Wet mounts, trichrome and Kinyoun acid-fast stained slides were prepared from the stool specimens. PCR was used for detecting the presence of Giardia spp., Entamoeba spp., Dientamoeba fragilis, Cryptosporidium spp., Blastocystis spp. Genotyping was realized by using Blastocystis hominis STS primers. RESULTS: In both study groups, any helminth eggs and other protozoa except Blastocystis spp. were not detected by microscopy and PCR, and also bacterial cultures were negative. Only stool microscopy was positive for Blastocystis spp. in 30% (9 of 30) of splenectomized patients and in 13% (4/30) of healthy controls. PCR for Blastocystis spp. was positive in 40% (12 of 30), B. hominis genotypes were 20% (6/30): STS1 in 10% (3/30) and STS3 in 10% (3/30) of splenectomized patients. In healthy controls Blastocystis spp. was 13% (4/30) by PCR and genotypes of B. hominis was not detected. The difference between the prevalence of Blastocystis spp. infection in splenectomized patients and control groups was statistically significant (p=0.020). Abdominal pain was the most frequent gastrointestinal symptom (p=0.019) among splenectomized patients positive for Blastocystis spp. CONCLUSION: In post-traumatic healthy splenectomized patients, Blastocystis spp. were found to be the most prevalent protozoa and may be responsible for the gastrointestinal disorders.