Solunum yolu örneklerinden saptanan rhinovirusların moleküler yöntemlerle tiplendirilmesi: Retrospektif bir çalışma
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Date
2023-07-12
Authors
Tuner, Beyza
Journal Title
Journal ISSN
Volume Title
Publisher
Bursa Uludağ Üniversitesi
Abstract
Rhinovirus (RV), Enterovirus (EV) cinsindeki Picornaviridae familyasında sınıflandırılır. RV-A, RV-B ve RV-C olmak üzere üç türden oluşur ve 170’in üzerinde RV alt tipi bulunmaktadır. Bu çalışmada, Bursa Uludağ Üniversitesi Tıbbi Mikrobiyoloji Anabilim Dalı PCR laboratuvarına gönderilen nazofaringeal sürüntü örneklerinde saptanan RV’lerin moleküler olarak alt tiplendirilmesini yapmak ve tiplerin mevsimsel dağılımını, yaş ve cinsiyet kategorileriyle olan ilişkisini belirlemek hedeflendi. V pozifliği saptanan, 0-90 yaş aralığındaki hastalara ait 80 adet örnekten viral RNA, Pure Link™ Viral RNA/DNA Mini Kit (ThermoFisher Scientific, ABD) kullanılarak ekstrakte edildi. Nükleik asit amplifikasyonu One Step RT-PCR Kiti (Qiagen, Almanya) kullanılarak gerçekleştirildi. 5’UTR’de korunmuş bölgeleri hedefleyen RV evrensel primerleri kullanılarak amplifiye edildi. Amplifikasyon işlemi sonrasında örnekler agaroz jel elektroforezine tabi tutularak pürifiye edildi. Pürifikasyon sonrası elde edilen PCR ürünleri Sanger dideoksi döngü dizileme metodu kullanılarak tekyönlü DNA dizi analizi yapıldı. Elde edilen diziler MEGA 11 programı kullanılarak değerlendirildi. Bu çalışmada, RV’lerin yıl boyu dolaşımda olduğu ve sonbaharda zirve yaptıkları tespit edildi. Çalışmaya dahil edilen 80 örnekte, RV-A 33 (%41,25), RV-B 8 (%10) ve RV-C 31 (%38,75) adet bulundu. Örneklerin üçü (%3,75) EV-D68 olarak tiplendirilirken, beş (%6,25) örnek tiplendirilemedi. RV-A için 16, RV-B için 3, RV C için 12 olmak üzere, toplam 31 farklı alt tip tespit edildi. RV-A, dört mevsimde de tespit edildi. RV-C enfeksiyonları sıklıkla sonbaharda meydana geldi (p<0,05). RV-B yalnızca ilkbahar ve sonbaharda tespit edildi. RV-A enfeksiyonlarının daha erken(p<0,05), RV-C enfeksiyonlarının ise daha geç yaşlarda görüldüğü tespit edildi(p<0,05). RV tiplerinin ve dağılımının, pandemi öncesi ülkemizde yapılan çalışmalarla benzerlik gösterdiği ve pandemiden etkilenmediği tespit edildi.
Rhinovirus (RV) is classified in the Picornaviridae family in the Enterovirus (EV) genus and consists of three species, RV-A, RV-B, and RV-C, and there are over 170 RV subtypes. This study aimed to make molecular subtyping of RVs detected in nasopharyngeal swab samples sent to Bursa Uludağ University Medical Microbiology Department PCR laboratory and to determine the seasonal distribution of types and their relationship with age and gender categories. Viral RNA was extracted from 80 samples of patients aged 0-90 years using the PureLink™ Viral RNA/DNA Mini Kit (ThermoFisher Scientific, USA). Nucleic acid amplification was performed using the OneStep RT-PCR Kit (Qiagen, Germany) by using RV universal primers targeting conserved regions in the 5'UTR. After the amplification process, the samples were purified after agarose gel electrophoresis. One-way DNA sequencing analysis of the PCR products obtained after purification was performed using the Sanger sequencing method. The obtained sequences were analyzed using the MEGA 11 program. In this study, RVs were found to circulate year-round and peak in the fall. In 80 samples, RV-A, RV-B, and RV-C were found in 31 (38.75%), 8 (10%), and 33 (41.25%) samples, respectively. Three samples (3.75%) were typed as EV-D68, while five (6.25%) samples could not be typed. Thirty-one subtypes were identified 16 for RV-A, 3 for RV-B, and 12 for RV-C. While RV-A was detected in all seasons, RV-C was frequent in autumn (p<0.05), and RV-B was detected in spring and autumn. We found that RV-A infections were seen at earlier ages (p<0.05), and RV-C infections were seen at later ages (p<0.05). It was found that RV types and distribution were similar to the studies conducted in our country before the pandemic, and types were not affected by the pandemic.
Rhinovirus (RV) is classified in the Picornaviridae family in the Enterovirus (EV) genus and consists of three species, RV-A, RV-B, and RV-C, and there are over 170 RV subtypes. This study aimed to make molecular subtyping of RVs detected in nasopharyngeal swab samples sent to Bursa Uludağ University Medical Microbiology Department PCR laboratory and to determine the seasonal distribution of types and their relationship with age and gender categories. Viral RNA was extracted from 80 samples of patients aged 0-90 years using the PureLink™ Viral RNA/DNA Mini Kit (ThermoFisher Scientific, USA). Nucleic acid amplification was performed using the OneStep RT-PCR Kit (Qiagen, Germany) by using RV universal primers targeting conserved regions in the 5'UTR. After the amplification process, the samples were purified after agarose gel electrophoresis. One-way DNA sequencing analysis of the PCR products obtained after purification was performed using the Sanger sequencing method. The obtained sequences were analyzed using the MEGA 11 program. In this study, RVs were found to circulate year-round and peak in the fall. In 80 samples, RV-A, RV-B, and RV-C were found in 31 (38.75%), 8 (10%), and 33 (41.25%) samples, respectively. Three samples (3.75%) were typed as EV-D68, while five (6.25%) samples could not be typed. Thirty-one subtypes were identified 16 for RV-A, 3 for RV-B, and 12 for RV-C. While RV-A was detected in all seasons, RV-C was frequent in autumn (p<0.05), and RV-B was detected in spring and autumn. We found that RV-A infections were seen at earlier ages (p<0.05), and RV-C infections were seen at later ages (p<0.05). It was found that RV types and distribution were similar to the studies conducted in our country before the pandemic, and types were not affected by the pandemic.
Description
Keywords
Rhinovirus, Dizi analizi, Moleküler epidemiyoloji, Sequence analysis, Molecular epidemiology
Citation
Tuner, B. (2023). Solunum yolu örneklerinden saptanan rhinovirusların moleküler yöntemlerle tiplendirilmesi: Retrospektif bir çalışma. Yayınlanmamış yüksek lisans tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.