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Journals / Ankara Üniversitesi Veteriner Fakültesi Dergisi / 2020 / Cilt: 67 - Sayı: 4

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

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

Yusuf DOĞRUER
Pages
349–355
Publication Date
2022-10-12
DOI
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⚡ TL;DR(Too Long; Didn't Read)

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

📌 3 Key Takeaways

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Objective & Hypothesis:One of the most challenging aspects in culture independent methods for foodborne pathogens’ detection is discrimination of dead and live microorganisms.
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Key Findings & Data: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.
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Conclusion & Impact: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
🔬Design:Research Article
📍Scope:200 samples
⚙️Variables:Domain Parameters

Abstract

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

Özet

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

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Cite This Article

DOĞRUER, Y. (2022). Determination of Vibrio parahaemolyticus in seafoods using directplate counting, quantitative loop-mediated isothermal amplificationand propidium monoazide-qLAMP. Ankara Üniversitesi Veteriner Fakültesi Dergisi, Cilt: 67 - Sayı: 4, 349–355. https://ajindex.com/articles/444469

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