Evagreen ve Taqman Tabanlı Gerçek Zamanlı RT-PCR Analizlerinin Validasyonu ve Karşılaştırılması: Apis Mellifera’da Black Queen Cell Virusunun Kantitatif Moleküler Teşhisi İçin Yeni Yaklaşımlar
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Dosyalar
Tarih
2023
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Black queen cell virus (BQCV), bal arılarında genelde subklinik enfeksiyon oluşturan ve
küresel dağılım gösteren yaygın bir virustur. BQCV‟nin diğer bazı bal arısı patojenleriyle (acute bee
paralysis virus, deformed wing virus, Nosema spp.) karşılıklı potansiyel sinerjik etkileşiminin ve bal
arısı biyolojisinde keşfedilmeyi bekleyen etkilerinin anlaşılması araştırmacıların ilgisini çekmiştir. Bu
çalışmanın amacı, erişkin arı ve larva-pupada BQCV‟nin tespiti için BQCV yapısal protein (SP-VP1)
ve yapısal olmayan protein (NSP-RdRp) genlerin amplifikasyonuna dayanan TaqMan prob ve
EvaGreen (EG) dye tabanlı kantitatif real-time reverse transcriptase PCR (qRT-PCR) analizlerinin
geliştirilmesi ve validasyonunun gerçekleştirilmesidir. Ayrıca real-time RT-PCR analizleri için yaygın
kullanılan EG dye ve TaqMan prob kimyasallarının analiz performansları karşılaştırıldı. Analizlerin
validasyonu için hazırlanan standart RNA alikotları ile oluşturulan standart eğrilere dayalı metot
etkinlik hesapları, lineer regresyon performansı ve güvenilirlik değerlerine dayalı doğruluk profilleri
bu analizlerin doğruluk, kesinlik, kabul edilebilirlik ve güvenilirlik açısından uygun olduğunu
kanıtladı. BQCV-SP TaqMan ve EG real-time qRT-PCR analizleri için çalışma aralığı sırasıyla 2,61-
7,61 log10 RNA kopyaları/reaksiyon ve 2,61-7,79 log10 RNA kopyaları/reaksiyon arasında belirlendi.
BQCV-NSP TaqMan ve EG real-time qRT-PCR analizleri için çalışma aralığı sırasıyla 6,83-11,83
log10 RNA kopyaları/reaksiyon ve 6,98-11,98 log10 RNA kopyaları/reaksiyon arasında tespit edildi.
Bu yeni analizleri kullanarak Akdeniz ve İç Anadolu Bölgelerinde doğal olarak enfekte olmuş
kovanlarda BQCV‟nin prevalansı ve viral yükü hesaplandı. BQCV‟nin prevalansı erişkin arı ve larvapupada sırasıyla %84 ve %44 bulundu, viral yükü ise validasyon sürecinde belirlenen analizlerin
çalışma aralığındaydı. Saha örneklerinin tamamı asemptomatik olduğundan, bu analizleri kullanarak
BQCV‟nin gizli ve açık enfeksiyonu arasındaki ayrımı tahmin edebilecek bir viral genom yükü
önerilemedi. Ancak larva-pupaların asemptomatik enfeksiyonunda BQCV-SP real-time qRT-PCR
analizi için ≤8,80 log10 RNA kopyalar/reaksiyon ve BQCV-NSP real-time qRT-PCR analizi için
≤11,80 log10 RNA kopyalar/reaksiyon gibi bir tanı eşik değeri bulundu. EG dye ve TaqMan prob
kimyasallarının analitik ve tanısal performans analizlerinde kıyaslanabilir sonuçlar elde edildi ve
hesaplanan Ct değerleri arasında istatistiksel olarak anlamlı bir fark bulunmadı (Wilcoxon Signed
Ranks Test 2-tailed). Böylece EG‟nin, Taqman proba benzer olarak, real-time RT-PCR analizleri için
sağladığı yüksek amplifikasyon etkinliği teyit edildi. Sonuç olarak BQCV‟nin tespiti için son derece
hassas ve analitik olarak spesifik olan bu yeni real-time qRT-PCR analizlerinin, BQCV‟nin etiyolojik
ve epidemiyolojik çalışmalarına katkı sağlayacağı ifade edilebilir.
Black queen cell virus (BQCV) is a common virus that causes covert infection in hives and has a global distribution. It is of interest to honey bee virologists to understand the potential synergistic interaction between BQCV and some other honeybee pathogens (acute bee paralysis virus, deformed wing virus, Nosema spp.) and to elucidate the unexplored effects of BQCV in honey bee biology. This study aims to develop and validate the TaqMan probe and EvaGreen (EG) dye-based real-time reverse transcriptase PCR (qRT-PCR) assays based on the amplification of the BQCV structural protein (SP-VP1) and non-structural protein (NSP-RdRp) genes for BQCV detection in adult honey bees and larva-pupa. Additionally, the analysis performances of commonly used EG and TaqMan chemicals for real-time RT-PCR analyses were compared. Method efficiency calculations based on standard curves created with standard RNA aliquots prepared for the validation of the analyses, linear regression performance, and accuracy profiles based on reliability values proved that these analyses were proper in terms of accuracy, precision, acceptability, and reliability. The operating range for BQCV-SP TaqMan and EG real-time qRT-PCR analyses was calculated from 2,61 to 7,61 log10 RNA copies/reaction and 2,61 to 7,79 log10 RNA copies/reaction, respectively. The operating range for BQCV-NSP TaqMan and EG real-time qRT-PCR analyses was calculated from 6,83 to 11,83 log10 RNA copies/reaction and 6,98 to 11,98 log10 RNA copies/reaction, respectively. Using these novel analyses, the prevalence and viral load of BQCV were calculated in naturally infected hives in the Mediterranean and Central Anatolia Regions. The prevalence of BQCV was found to be 84% and 44% in adult honey bees and larvae-pupae, respectively. BQCV viral load was within the operating range of the analyses determined during the validation. All of these samples had the covert infection. Therefore, a viral genome load that could predict the distinction between covert and overt infection of BQCV using the analyses could not be proposed. However, a diagnostic threshold value of ≤8,80 log10 RNA copies/reaction for BQCV real-time qRT-PCR analysis and ≤11,80 log10 RNA copies/reaction for BQCV real-time qRT-PCR analysis was found in the covert infection of larvaepupae. Comparable results were obtained in analytical and diagnostic performance analyses of the EG dye and TaqMan probe chemicals, and no statistically (Wilcoxon Signed Ranks Test 2-tailed) significant difference was found between the calculated Ct values. Thus, the high amplification efficiency of the EG dye for RT-PCR analyses was confirmed, similar to the Taqman probe. In conclusion, highly sensitive and analytically specific for the detection of BQCV, these novel real-time qRT-PCR assays will benefit the etiological and epidemiological studies of BQCV.
Black queen cell virus (BQCV) is a common virus that causes covert infection in hives and has a global distribution. It is of interest to honey bee virologists to understand the potential synergistic interaction between BQCV and some other honeybee pathogens (acute bee paralysis virus, deformed wing virus, Nosema spp.) and to elucidate the unexplored effects of BQCV in honey bee biology. This study aims to develop and validate the TaqMan probe and EvaGreen (EG) dye-based real-time reverse transcriptase PCR (qRT-PCR) assays based on the amplification of the BQCV structural protein (SP-VP1) and non-structural protein (NSP-RdRp) genes for BQCV detection in adult honey bees and larva-pupa. Additionally, the analysis performances of commonly used EG and TaqMan chemicals for real-time RT-PCR analyses were compared. Method efficiency calculations based on standard curves created with standard RNA aliquots prepared for the validation of the analyses, linear regression performance, and accuracy profiles based on reliability values proved that these analyses were proper in terms of accuracy, precision, acceptability, and reliability. The operating range for BQCV-SP TaqMan and EG real-time qRT-PCR analyses was calculated from 2,61 to 7,61 log10 RNA copies/reaction and 2,61 to 7,79 log10 RNA copies/reaction, respectively. The operating range for BQCV-NSP TaqMan and EG real-time qRT-PCR analyses was calculated from 6,83 to 11,83 log10 RNA copies/reaction and 6,98 to 11,98 log10 RNA copies/reaction, respectively. Using these novel analyses, the prevalence and viral load of BQCV were calculated in naturally infected hives in the Mediterranean and Central Anatolia Regions. The prevalence of BQCV was found to be 84% and 44% in adult honey bees and larvae-pupae, respectively. BQCV viral load was within the operating range of the analyses determined during the validation. All of these samples had the covert infection. Therefore, a viral genome load that could predict the distinction between covert and overt infection of BQCV using the analyses could not be proposed. However, a diagnostic threshold value of ≤8,80 log10 RNA copies/reaction for BQCV real-time qRT-PCR analysis and ≤11,80 log10 RNA copies/reaction for BQCV real-time qRT-PCR analysis was found in the covert infection of larvaepupae. Comparable results were obtained in analytical and diagnostic performance analyses of the EG dye and TaqMan probe chemicals, and no statistically (Wilcoxon Signed Ranks Test 2-tailed) significant difference was found between the calculated Ct values. Thus, the high amplification efficiency of the EG dye for RT-PCR analyses was confirmed, similar to the Taqman probe. In conclusion, highly sensitive and analytically specific for the detection of BQCV, these novel real-time qRT-PCR assays will benefit the etiological and epidemiological studies of BQCV.
Açıklama
Anahtar Kelimeler
Bal arısı, BQCV, EvaGreen dye, real-time qRT-PCR, TaqMan prob, Honey bee
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Öz, M. E., (2023). Evagreen ve Taqman Tabanlı Gerçek Zamanlı RT-PCR Analizlerinin Validasyonu ve Karşılaştırılması: Apis Mellifera’da Black Queen Cell Virusunun Kantitatif Moleküler Teşhisi İçin Yeni Yaklaşımlar. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
