Microfluidic chip fabrication in three-dimensional (3D) printer combined with loop-mediated isothermal amplification (LAMP) for detection of Salmonella spp.

Öz

Rapid, reliable and sensitive detection of Salmonella in food and environmental sources is essential to protect public health. LAMP method has been preferred frequently in recent years because it is a method that can be applied without the need for special devices and experienced personnel. Combining LAMP with microfluidic devices is an alternative for rapid diagnostic testing where resources are limited. Aim of this study was to create a microfluidic chip using a biocompatible PLA filament in a three-dimensional printer and to be combined with the loop-mediated isothermal amplification method for the detection of Salmonella spp Microfluidic chips were designed using the CATIA V5 program (chip dimensions 15x12.5x2.4 mm, channel diameter 850 µm) and fabricated on a 3D printer using PLA filament. InvA gene was selected for Salmonella specific LAMP reactions. Dry heat block was used for microfluidic chips and LAMP protocol was 30 min. at 65℃. Sensitivity, specificity, PPV and NPV measurements of the test were performed. Among the 25 positive samples that were found to be Salmonella positive by the culture method, only one sample showed a false negative result with the LAMP reaction applied in the microcentrifuge tube, while two samples were detected as false negative in the LAMP reaction applied on the microfluidic chip. As a conclusion, Our data has shown us that LAMP reactions and microfluidic chip technology can be combined, and that three-dimensional printers can be used during the production of these microfluidic chips.

Anahtar Kelimeler

• LAMP
• Salmonella
• Microfluidic
• 3D

Salmonella spp. tespiti için ilmiğe dayalı izotermal amplifikasyon (LAMP) ile kombine üç boyutlu (3B) yazıcıda mikroakışkan çip imalatı

Öz

Gıda ve çevresel kaynaklarda Salmonella'nın hızlı, güvenilir ve hassas tespiti, halk sağlığını korumak için çok önemlidir. LAMP yöntemi özel cihazlara ve deneyimli personele ihtiyaç duymaksızın uygulanabilecek bir yöntem olduğu için son yıllarda sıklıkla tercih edilmektedir. LAMP’ın, mikroakışkan cihazlarla kombine edilmesi sınırlı kaynakların olduğu durumlarda, hızlı tanı testleri için bir alternatifdir. Salmonella spp. tespiti için, üç boyutlu yazıcıda biyouyumlu PLA filament kullanılarak mikroakışkan çip oluşturularak, ilmiğe dayalı izotermal amplifikasyon yöntemi ile kombine edilmesi amaçlanmıştır. Mikroakışkan çipler CATIA V5 programı kullanılarak tasarlandı (Çip boyutları 15x12.5x2.4 mm, kanal çapı 850 µm) ve PLA filament kullanılarak 3B yazıcıda üretildi. Salmonella spesifik LAMP reaksiyonları için InvA geni seçildi. Mikroakışkan çipler için kuru ısı bloğu kullanıldı ve LAMP protokolü 65℃’da 30 dak.’ydı. Testin duyarlılık, özgüllük, PPD ve NPD ölçümleri gerçekleştirildi. Kültür yöntemi ile Salmonella pozitif saptanan 25 pozitif numune arasında, mikrosantrifüj tüpünde uygulanan LAMP reaksiyonu ile sadece bir numunede yalancı negatif sonuc saptanmışken, mikroakışkan çip üstünde uygulanan LAMP reaksiyonunda ise iki numune yalancı negatif olarak tespit edildi. Sonuç olarak; verilerimiz LAMP reaksiyonları ile mikroakışkan çip teknolojisinin birleştirilebileceğini, üç boyutlu yazıcıların bu mikroakışkan çiplerin üretimi sırasında kullanılabileceğini bize göstermiştir.

Anahtar Kelimeler

• LAMP
• Salmonella
• Mikroakışkan
• 3B

Kaynakça

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