Evaluation of a Chelex 100-based DNA extraction procedure for morphologically identified adult cyathostomins

Siatostominlar (Nematoda: Strongylida: Cyathostominae) otlarning keng tarqalgan oshqozon-ichak nematodalari bo‘lib, taksonomik jihatdan xilma-xil guruhni tashkil etadi. Tadqiqotning maqsadi morfologik jihatdan identifikatsiya qilingan alohida voyaga yetgan siatostominlardan DNK ajratib olishning Chelex 100 asosidagi moslashtirilgan usulini baholash hamda olingan DNK miqdori va spektrofotometrik ko‘rsatkichlarini aniqlashdan iborat bo‘ldi. O‘n bitta voyaga yetgan siatostomin morfologik jihatdan identifikatsiya qilindi va ularning har biriga alohida ishlov berildi. DNK konsentratsiyasi hamda A260/A280 va A260/A230 optik zichlik nisbatlari mikrohajmli spektrofotometriya usulida aniqlandi. Barcha 11 ta ekzemplyardan o‘lchash mumkin bo‘lgan miqdorda nuklein kislota olindi. DNK konsentratsiyasi 1,9–106,6 ng/mkl oralig‘ida bo‘lib, mediana 12,5 ng/mkl, o‘rtacha qiymat esa 29,9 ng/mkl bo‘ldi. A260/A280 nisbati 1,80–6,33 oralig‘ida o‘zgardi; mediana 1,97 bo‘lib, o‘n bitta ekstraktdan oltitasida bu ko‘rsatkich 1,80–2,00 oralig‘ida qayd etildi. Ekstraktlar qo‘shimcha ravishda PZR usulida tekshirildi. Tayyorlangan DNK namunalarida amplifikatsiya kuzatildi, bu esa amplifikatsiyaga yaroqli DNK matritsasi mavjudligini tasdiqladi. Chelex 100/proteinaza K usuli alohida ishlov berilgan, morfologik jihatdan identifikatsiya qilingan voyaga yetgan siatostominlardan kolonkada tozalash va organik erituvchilarni qo‘llamasdan, namunani ko‘chirish bosqichlari soni cheklangan holda DNK olish imkonini berdi. Ushbu metoddan alohida siatostominlarni morfologik identifikatsiya qilish va molekulyar-genetik tahlilni birlashtirgan tadqiqotlarda namunani tayyorlashning oddiy usuli sifatida foydalanish mumkin.

“PARAZITAR KASALLIKLAR DIAGNOSTIKASIDA MOLEKULYAR GENETIK USULLARNI QO‘LLASH, DAVOLASH VA PROFILAKTIKASINING ZAMONAVIY JIHATLARI”

MAVZUSIDAGI XALQARO ILMIY-AMALIY KONFERENSIYA · SAMARQAND, 2026-YIL 9–10-OKTYABR

Ingliz tilida UDK: 619:616.995.1:577.2 B. 17–19

EVALUATION OF A CHELEX 100-BASED DNA EXTRACTION PROCEDURE FOR MORPHOLOGICALLY IDENTIFIED ADULT CYATHOSTOMINS

Demkina O.V. 1, Kotelnikov D.D. 2 Far Eastern State Agrarian University, Blagoveshchensk, Russia Institute for Agrarian Studies, HSE University, Moscow, Russia

DOI https://doi.org/10.66073/ReFocus-Conf/260902

I QT I B O S U C H U N · Д Л Я Ц И Т И Р О В А Н И Я · FO R C I TAT I O N

Demkina O.V., Kotelnikov D.D. Evaluation of a Chelex 100-based DNA extraction procedure for morphologically identified adult cyathostomins // Parazitar kasalliklar diagnostikasida molekulyar genetik usullarni qo‘llash, davolash va profilaktikasining zamonaviy jihatlari: xalqaro ilmiy-amaliy konferensiya materiallari (Samarqand, 9–10-oktyabr 2026-y.). – Samarqand, 2026. – B. 17–19. – DOI: https://doi.org/10.66073/ReFocus-Conf/260902

Research Focus xalqaro ilmiy jurnali hamkorligida Research Focus International Scientific Journal · refocus.uz

UDC: 619:616.995.1:577.2 EVALUATION OF A CHELEX 100-BASED DNA EXTRACTION PROCEDURE FOR MORPHOLOGICALLY IDENTIFIED ADULT CYATHOSTOMINS Demkina O.V.¹, Kotelnikov D.D.² ¹ Far Eastern State Agrarian University 86 Polytechnicheskaya St., Blagoveshchensk, Amur Region, Russia ² Institute for Agrarian Studies, HSE University 11 Pokrovsky Boulevard, Moscow, 109028, Russia. Cyathostomins (Nematoda: Strongylida: Cyathostominae) are common gastrointestinal nematodes of horses and comprise a taxonomically diverse group. Morphological identification of adult specimens remains important for species determination, whereas molecular approaches provide additional possibilities for taxonomic verification and analysis of genetic variation, including polymorphisms associated with anthelmintic resistance [1, 2]. Molecular assays for small strongyles require DNA of sufficient quality for amplification; therefore, a simple procedure that permits individual morphologically identified specimens to be processed separately is useful for combined morphological and molecular studies. Coles et al. [1] also emphasized that the integrity of DNA used in molecular assays may be assessed by amplification of independent gene sequences. The aim of the study was to evaluate an adapted Chelex 100-based procedure for DNA extraction from individual morphologically identified adult cyathostomins and to assess the yield and spectrophotometric characteristics of the obtained DNA. PCR amplification was used only as an additional functional test of DNA suitability for subsequent molecular analysis. Eleven adult cyathostomins were morphologically identified and processed individually. Each specimen was transferred into 50 µL of a 5% Chelex 100 m/w, and 2 µL of proteinase K (20 mg/mL) was added. Samples were incubated at 56°C for 1 h and subsequently heated at 99°C for 8 min. After centrifugation at 15,000 × g for 3 min, the supernatant containing DNA was collected without disturbing the Chelex resin. Before PCR, DNA extracts were diluted 1:10 with nuclease-free water. DNA concentration and absorbance ratios A260/A280 and A260/A230 were determined by microvolume spectrophotometry. Measurable nucleic acid was obtained from all 11 specimens. DNA concentrations ranged from 1.9 to 106.6 ng/µL, with a median of 12.5 ng/µL and a mean of 29.9 ng/µL. The A260/A280 ratio ranged from 1.80 to 6.33; the median value was 1.97, and six of the eleven extracts had values between 1.80 and 2.00. The highest A260/A280 ratio (6.33) occurred in the extract with the lowest DNA concentration (1.9 ng/µL), indicating that absorbance ratios should be interpreted cautiously when absolute absorbance values are low. A260/A230 values were highly variable and were therefore not used as the principal criterion of extract suitability. The extracts were additionally tested by PCR. Amplification was obtained from the prepared DNA samples, confirming the presence of amplifiable template.

In the two PCR runs, the amplification curves crossed the preset fluorescence threshold at approximately cycles 14–30 and 20–30, respectively. These values were used only as a functional characteristic of the extracts and were not interpreted quantitatively. Detailed evaluation of amplification performance and taxon-specific PCR was outside the scope of the present study. The Chelex 100/proteinase K procedure provided DNA from individually processed, morphologically identified adult cyathostomins without column purification or organic solvents and with a limited number of sample-transfer steps. This is relevant when individual parasite specimens must remain analytically independent for subsequent molecular characterization. PCR amplifiability provided a more direct indication of downstream suitability than spectrophotometric ratios alone, particularly for low-concentration extracts. In conclusion, the adapted Chelex 100-based procedure allowed DNA to be recovered from all examined adult cyathostomins and produced templates suitable for subsequent PCR amplification. The method can be used as a simple preparatory approach in studies combining morphological identification of individual cyathostomins with molecular genetic analysis.