Ekinezya Ürünlerine Hile Amaçlı Karıştırılan Parthenium integrifolium’un Döngü Aracılı İzotermal Amplifikasyon Yöntemiyle Tespiti

Öz

Amaç: Ekinezya, tıbbi amaçlarla dünya çapında yaygın olarak kullanılan bir bitkidir. Ekinezya ürünlerinin önemli bir pazar büyüklüğüne ulaşması, bu ürünlere yönelik hile amaçlı uygulamalarla ilgili endişelerin de artmasına yol açmıştır. Bitkisel ticari ürünlerin test edilmesi için kullanılan geleneksel yöntemler genellikle bitki türlerinin ayırt edici morfolojik özelliklerine dayanmaktadır. Bu nedenle toz haline getirilmiş ya da endüstriyel işlemlere tabi tutulmuş bitki materyallerinde işe yaramamaktadır. Bu çalışmada, Ekinezya ürünlerine hile amaçlı karıştırıldığı bilinen Parthenium integrifolium türünün tespitini sağlayan döngü aracılı izotermal amplifikasyon (LAMP) temelli bir yöntemin geliştirilmesi ve test edilmesi amaçlanmıştır. Materyal ve Yöntem: Çalışma kapsamında, tıbbi amaçlı kullanılan Echinacea purpurea, Echinacea angustifolia ve Echinacea pallida türleri ve Parthenium integrifolium bitkisi temin edilmiş ve bu bitkilerden DNA izolasyonu yapılmıştır. Veri tabanlarındaki ham Parthenium integrifolium DNA dizileri analiz edilerek hedef gen bölgeleri tespit edilmiştir. Daha sonra bu hedef bölgeleri çoğaltacak spesifik LAMP primer dizileri tasarlanmış ve optimizasyonlar gerçekleştirilmiştir. Araştırma Bulguları: Yapılan çalışmalar sonucunda, 65 oC sabit sıcaklıkta uygulanan ve hile amaçlı karıştırılan bitki materyali varlığını reaksiyon karışımındaki renk değişimi sayesinde, ek bir analize gerek kalmadan tespit edebilen DNA temelli bir yöntem geliştirilmiştir. Geliştirilen yöntem için P. integrifolium tespit sınırı 0,5 ng, özgüllük ise %100 olarak saptanmıştır. Bu yöntemle İstanbul’da temin edilen 50 ticari Echinacea ürünü test edilmiş ve örneklerin hiçbirinde P. integrifolium varlığına rastlanmamıştır. Sonuç: Bu çalışmada, Echinacea ürünlerinde Parthenium integrifolium gibi tağşiş unsurlarının varlığını tespit etmeye yönelik, LAMP temelli yeni bir moleküler yöntem geliştirilmiştir.

Anahtar Kelimeler

• Ekinezya
• Parthenium integrifolium
• LAMP
• tıbbi bitkiler
• DNA

Detection of Parthenium integrifolium Used for Adulteration in Echinacea Products by a Loop Mediated Isothermal Amplification Method

Abstract

Objective: Echinacea is a plant widely used worldwide for medical purposes. The fact that Echinacea products have reached a significant market size has also led to increased concerns about adulteration of these products. Traditional methods for testing herbal commercial products are often based on the differential morphological characteristics of the plant species. Therefore, plant materials that have been powdered or subjected to industrial processing cannot be tested using these methods. The aim of this study was to develop and test a loop-mediated isothermal amplification (LAMP)-based method that detects the Parthenium integrifolium species which is known to be used for adulteration of Echinacea products. Materials and Methods: Echinacea purpurea, Echinacea angustifolia, Echinacea pallida and Parthenium integrifolium plants were obtained and used for DNA isolations. By analysis of raw Parthenium integrifolium DNA sequences in the public databases, specific LAMP primer sequences were designed to amplify targeted gene regions and then reaction optimizations were performed. Results: As a result of the study, a DNA-based method which is applied at a constant temperature of 65 °C and detects the presence of plant material mixed for adulteration purposes, by means of the color change in the reaction mixture, without the need for an additional analysis has been developed. The method had a detection limit of 0.5 ng for Parthenium integrifolium and a specificity of 100%. 50 commercial Echinacea products from Istanbul were tested and the results showed no presence of Parthenium integrifolium in the products. Conclusion: In this study, a new molecular method based on LAMP was developed to detect the presence of adulterants such as Parthenium integrifolium in Echinacea products.

Keywords

• Echinacea
• Parthenium integrifolium
• medicinal plants
• isothermal amplification
• DNA

Kaynakça

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