Brucella dna'sının teshisi amacıyla altın nanoparçacıklarının kullanıldıgı aunps-prob nanoteshis metodunun gelistirilmesi
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2017
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info:eu-repo/semantics/openAccess
Özet
Brusellozis; hayvanlarda genital organlara yerleşerek yavru atmalara ve infertiliteye neden olmakta, ülkelerin ekonomilerini olumsuz yönde etkilemektedir. Son yıllarda, bakteriyel hastalıkların teşhisi amacıyla altın nano parçacıklarının (AuNPs) kullanıldığı AuNPs-prob nanoteşhis yöntemleri geliştirilmiştir. Bu metot ile en az miktardaki genomik DNA’lar bile kullanılan nano parçacıklardan dolayı başarı ile tespit edilmektedir. Fakat Brucella enfeksiyonlarının hızlı nano teşhisi amacıyla geliştirilmiş herhangi bir AuNPs-prob nanoteşhis metodu bulunmamaktadır. Bu araştırma projesi ile, ülkemiz hayvancılığına ciddi zararlar veren brusellozisin direk teşhisi amacıyla hızlı, güvenilir, yüksek hassasiyete sahip ve DNA amplifikasyonu gerektirmeyen AuNPs-prob nanoteşhis yönteminin geliştirilmesi amaçlandı. Bu amaçla, Brucella cinsine özgü 16S-23S rRNA veya omp2 gen bölgeleri ile hibridize olabilecek 3 prob dizayn edildi, elde edilen problar altın nanoparçacıklarına tutturularak AuNPs-prob nanoteşhis metodu geliştirildi. Metodun spesifite, sensitivite ve tekraralanabilirliğini test etmek için 50 adet koyun aborte fetüs mide içeriği, 50 adet inek aborte fetüs mide içeriği, 50 adet inek süt örneği ve 50 adet koyun süt örneği kullanıldı. Örnekler Brucella varlığı yönünden kültür ve PZR yöntemleri ile incelendi. Brucella yönünden pozitif bulunmuş atık fetüs örneklerinin ait olduğu 12 sürüden toplanmış 410 kan örneği RBPT ve SAT testleri ile incelendi ve sonuçları AuNPs-prob nanoteşhis yöntem sonuçları ile karşılaştırıldı. Toplam 200 saha örneğinin 28’i kültür, 30’u PZR ve 25’i AuNPs-prob nanoteşhis yöntemi ile Brucella yönünden pozitif olarak bulundu. Geliştirilen yöntemin belirleyebildiği en az DNA miktarı B. melitensis, B. aborus ve B. canis referans suşları için sırasıyla 93, 101 ve 88 ng olarak tespit edildi. AuNPs-prob nanoteşhis metodunun sensitivitesi %89.3, spesifitesi %100 ve tekrarlanabilirliği %100 olarak hesap edildi. Serolojik olarak Brucella pozitif olan 12 sürüden toplanmış 12 aborte fetüs mide içeriğinin 11’inde Brucella DNA’sı AuNPs-prob nanoteşhis yöntemi ile tespit edildi. Sonuç olarak, brusellozisin teşhisine yönelik AuNPs-prob nanoteşhis yöntemi bu proje kapsamında geliştirildi. Bu yöntem ile brusellozisin hem insanlarda hem de hayvanlarda hızlı, ucuz, güvenli ve en önemlisi küçük laboratuarlarda pahalı PZR vb cihazlara gereksinim duyulmadan teşhisi sağlanacaktır.
Brucellosis causes infertility and abortion by invasion to genital organs of animals and negatively affects economies of countries. AuNPs-probe nanodiagnostic methods in which gold nano particules are used have recently been developed for detection of bacterial diseases. By this method a successful detection of the least amounts of genomic DNA can be possible by means of these nano particles. However, there is no AuNPs-probe based method developed for the rapid nanodiagnosis of Brucellosis yet. In order to direct diagnose Brucellosis, causes serious damage to the Country's livestock an AuNPs-probe nanodiagnosis method which would be fast, reliable, highly sensitive and not required for a DNA amplification step is aimed by this research project. For this purpose, 3 DNA probes specific for Brucella 16S-23S rRNA or omp2 gene regions were designed. They were then attached to gold nano particles (AuNPs) and an AuNPs-probe nanodiagnosis assay was developed. Fifty of each of samples from sheep aborted fetus stomach contents, cattle aborted fetus stomach contents, cattle milk and sheep milk were used to measure specificity, sensitivity and repeatability of the method. The samples were examined by culture and PCR assays. A total of 410 blood samples collected from 12 herds that were already found positive for Brucella presence were tested by RBPT and SAT. The results by these serological tests were compared with results of AuNPs-probe nanodiagnosis assay. Among 200 field samples tested, 28, 30 and 25 were found to be positive for Brucella by culture, PCR and AuNPs-prob nanodiagnosis methods, respectively. Minimum detection limits of the developed method were defined as 93, 101 and 88 ng DNAs for B. melitensis, B. aborus and B. canis reference strains, respectively. Sensitivity, specificity and repeatability of the developed assay were 89.3%, 100%, and 100%, respectively. All the twelve herds were serologically found Brucella positive however, 11 aborted fetus stomach contents taken from of these serologically positive flocks were shown to have Brucella DNA by AuNPs-prob nanodiagnosis method. In conclusion, an AuNPs-prob nanodiagnosis method was developed for diagnose Brucellosis. By this method, Brucellosis in man and animals can be diagnosed quicker, cheaper, safer than used to be and more importantly in small sized laboratories and without a need for expensive equipment like those for PCR.
Brucellosis causes infertility and abortion by invasion to genital organs of animals and negatively affects economies of countries. AuNPs-probe nanodiagnostic methods in which gold nano particules are used have recently been developed for detection of bacterial diseases. By this method a successful detection of the least amounts of genomic DNA can be possible by means of these nano particles. However, there is no AuNPs-probe based method developed for the rapid nanodiagnosis of Brucellosis yet. In order to direct diagnose Brucellosis, causes serious damage to the Country's livestock an AuNPs-probe nanodiagnosis method which would be fast, reliable, highly sensitive and not required for a DNA amplification step is aimed by this research project. For this purpose, 3 DNA probes specific for Brucella 16S-23S rRNA or omp2 gene regions were designed. They were then attached to gold nano particles (AuNPs) and an AuNPs-probe nanodiagnosis assay was developed. Fifty of each of samples from sheep aborted fetus stomach contents, cattle aborted fetus stomach contents, cattle milk and sheep milk were used to measure specificity, sensitivity and repeatability of the method. The samples were examined by culture and PCR assays. A total of 410 blood samples collected from 12 herds that were already found positive for Brucella presence were tested by RBPT and SAT. The results by these serological tests were compared with results of AuNPs-probe nanodiagnosis assay. Among 200 field samples tested, 28, 30 and 25 were found to be positive for Brucella by culture, PCR and AuNPs-prob nanodiagnosis methods, respectively. Minimum detection limits of the developed method were defined as 93, 101 and 88 ng DNAs for B. melitensis, B. aborus and B. canis reference strains, respectively. Sensitivity, specificity and repeatability of the developed assay were 89.3%, 100%, and 100%, respectively. All the twelve herds were serologically found Brucella positive however, 11 aborted fetus stomach contents taken from of these serologically positive flocks were shown to have Brucella DNA by AuNPs-prob nanodiagnosis method. In conclusion, an AuNPs-prob nanodiagnosis method was developed for diagnose Brucellosis. By this method, Brucellosis in man and animals can be diagnosed quicker, cheaper, safer than used to be and more importantly in small sized laboratories and without a need for expensive equipment like those for PCR.
Açıklama
Aras, Zeki ( Aksaray, Yazar )
Anahtar Kelimeler
Brucella, Brusellozis, Altın Nano Parçacık, Nanoteşhis, Aunps-Prob Nanoteşhis Metodu, Brucellosis, Gold Nano Particles, Nanodiagnosis, Aunps-Probe Nanodiagnosis Assay
