Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP

DOGRUER, YUSUF

doi:10.33988/auvfd.603868

Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP

Yusuf DOĞRUER (Selçuk Üniversitesi, Veteriner Fakültesi, Gıda Hijyeni ve Teknolojisi Bölümü, Konya, Türkiye)

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Yıl: 2020 Cilt: 67 Sayı: 4 Sayfa Aralığı: 349 - 355 Metin Dili: İngilizce DOI: 10.33988/auvfd.603868 İndeks Tarihi: 14-06-2021

Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP

Öz:

One of the most challenging aspects in culture independent methods for foodborne pathogens’ detection is discrimination of dead and live microorganisms. This study aimed to determine the Vibrio parahaemolyticus in seafoods via direct plate counting (DPC) and toxR-based quantitative loop-mediated isothermal amplification (qLAMP) and to discriminate dead and live cells using propidium monoazide (PMA)-qLAMP. A total of 200 samples including finfishes (n= 100) and shrimps (n= 100), representing the Mediterranean, Black and Aegean sea were collected from supermarkets and fish markets of Konya-Turkey. qLAMP was performed in a Real-Time Turbidimeter and the time threshold (tt) values were yielded in 60 minutes. On DPC, the colonies grown on TCBS Agar was further confirmed by conventional PCR based from gyrB1 gene of Vibrio spp. and toxR gene of V. parahaemolyticus. Virulence property of the isolates were determined by tdh based qLAMP. The detection limit of the qLAMP was 1.2×104 CFU/g in artificially contaminated seafoods. DPC, qLAMP and PMA-qLAMP detected V. parahaemolyticus in 8 (4%), 12 (6%) and 12 (6%) samples, respectively. The CFUs of V. parahaemolyticus detected in qLAMP (5.96±0.10 log10 CFU/ml) and PMAqLAMP (4.71±0.13 log10 CFU/ml) were higher than those of DPC (1.99±0.44 log10 CFU/ml) (P<0.05). The mean tt reduction in PMA treated samples was 1.25±0.38 log10 CFU/sample. The tdh gene was not detected in any of the isolates. In conclusion, the toxR-based PMA-qLAMP method could be an alternative to be used more widely and effective assay for the quantification of live V. parahaemolyticus in seafoods.

Anahtar Kelime:

Deniz ürünlerinde Vibrio parahaemolyticus’un direkt kültür yöntemi, kantitatif ilmiğe dayalı izotermal amplifikasyon ve propidium monoazide-qLAMP ile belirlenmesi

Öz:

Gıda kaynaklı patojenlerin kültür bağımsız yöntemlerle tespitinde en zorlu hususlardan biri ölü ve canlımikroorganizmaların ayırt edilmesidir. Bu çalışmada, deniz ürünlerinde V. parahaemolyticus varlığının direkt kültür yöntemi (DPC)ve toxR bazlı kantitatif ilmiğe dayalı izotermal amplifikasyon (qLAMP) ve canlı-ölü hücre ayrımı için propidium monoazide (PMA)- qLAMP yoluyla belirlenmesi amaçlandı. Akdeniz, Karadeniz ve Ege denizlerini temsil eden balık (n= 100) ve karides (n= 100) olmaküzere toplam 200 örnek, Konya-Türkiye’de bulunan süpermarketler ve balık hallerinden toplandı. Real-Time Turbidimetredegerçekleştirilen qLAMP reaksiyonunda time threshold (tt) değerleri, 60 dakika içinde elde edildi. DPC metodunda TCBS Agar'dagelişen kolonilerde Vibrio spp.’nin doğrulanması için gyrB1 genini, V. parahaemolyticus’un doğrulanması için de toxR geniniamplifiye eden konvansiyonel PCR yöntemi kullanıldı. İzolatların virülans özelliğinin belirlenmesinde tdh bazlı qLAMP kullanıldı.qLAMP'ın deneysel olarak kontamine edilen deniz ürünlerinde tespit limiti 1.2 x 104 CFU/g olarak belirlendi. DPC, qLAMP ve PMA qLAMP yöntemleri ile sırasıyla 8 (%4), 12 (%6) ve 12 (%6) örnekte V. parahaemolyticus tespit edildi. qLAMP (5.96 ± 0.10 log10 KOB/ml) ve PMA-qLAMP (4.71±0.13 log10 KOB/ml) yöntemlerinde tespit edilen V. parahaemolyticus sayılarının DPC yönteminegöre (1.99 ± 0.44 log10 KOB/ml) daha yüksek olduğu gözlemlendi (P<0.05). PMA uygulanan örneklerde ortalama tt azalması, 1.25 ±0.38 log10 KOB/örnek olarak tespit edildi. İzolatların hiçbirinde tdh geni tespit edilmedi. Sonuç olarak; toxR-bazlı PMA-qLAMPyönteminin, deniz ürünlerinde canlı V. parahaemolyticus'un kantitatif tespitinde daha yaygın ve etkin kullanılabilecek alternatif biryöntem olabileceği düşünülmektedir

Anahtar Kelime:

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

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APA	DOGRUER Y (2020). Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. , 349 - 355. 10.33988/auvfd.603868
Chicago	DOGRUER YUSUF Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. (2020): 349 - 355. 10.33988/auvfd.603868
MLA	DOGRUER YUSUF Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. , 2020, ss.349 - 355. 10.33988/auvfd.603868
AMA	DOGRUER Y Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. . 2020; 349 - 355. 10.33988/auvfd.603868
Vancouver	DOGRUER Y Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. . 2020; 349 - 355. 10.33988/auvfd.603868
IEEE	DOGRUER Y "Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP." , ss.349 - 355, 2020. 10.33988/auvfd.603868
ISNAD	DOGRUER, YUSUF. "Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP". (2020), 349-355. https://doi.org/10.33988/auvfd.603868

APA	DOGRUER Y (2020). Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 67(4), 349 - 355. 10.33988/auvfd.603868
Chicago	DOGRUER YUSUF Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. Ankara Üniversitesi Veteriner Fakültesi Dergisi 67, no.4 (2020): 349 - 355. 10.33988/auvfd.603868
MLA	DOGRUER YUSUF Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. Ankara Üniversitesi Veteriner Fakültesi Dergisi, vol.67, no.4, 2020, ss.349 - 355. 10.33988/auvfd.603868
AMA	DOGRUER Y Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. Ankara Üniversitesi Veteriner Fakültesi Dergisi. 2020; 67(4): 349 - 355. 10.33988/auvfd.603868
Vancouver	DOGRUER Y Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. Ankara Üniversitesi Veteriner Fakültesi Dergisi. 2020; 67(4): 349 - 355. 10.33988/auvfd.603868
IEEE	DOGRUER Y "Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP." Ankara Üniversitesi Veteriner Fakültesi Dergisi, 67, ss.349 - 355, 2020. 10.33988/auvfd.603868
ISNAD	DOGRUER, YUSUF. "Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP". Ankara Üniversitesi Veteriner Fakültesi Dergisi 67/4 (2020), 349-355. https://doi.org/10.33988/auvfd.603868