Öz
In the past three decades, as an alternative to PCR (polymerase chain reaction) new diagnostic techniques like LAMP (loop mediated isothermal amplification) whereby target DNA can be amplified under isothermal conditions without using thermocycler have been developed. The LAMP method allows the synthesis of large amounts of DNA in a short time with high specificity and rapid and easy detection of generated products. In this study, specificity and sensitivity of LAMP method was evaluated for the detection of T. annulata in acute infected and/or carriers cattle using primer pair specifically designed to amplify merozoite surface antigen gene (Mero1), 30 kDa major merozoite surface antigen gene (Tams-1) and cytochrome b gene of T.annulata. Primer pairs with highest sensitivity were used to evaluate the applicability of LAMP to the field samples. Two LAMP primers (CYTOB1 and CYTOB341) targeting cytochrome b gene specifically amplified DNA of different T. annulata isolates successfully while no amplification was seen in other species DNAs and BL20. CYTOB1 primers detected T. annulata Ankara / D7 DNA up to 2 fg, however the detection limit of CYTOB341 was 10 fold lower. The sensitivity of CYTOB1 LAMP assay was same with F3/B3 PCR, however when compared with that of cytob1 PCR a 10fold lower sensitivity was found. The LAMP product was confirmed by restriction digestion and sequencing. Results obtained from this study indicated that none of the designed primer pairs specific to target genes (Tams-1 and Mero1), except cytochrome b gene was able to specifically and sensitively detect different isolates of T. annulata. Consequently, it was shown that LAMP method using CYTOB1 primers is less effective than the cytob1 PCR in terms of detecting T. annulata in the field samples
Anahtar Kelimeler
Kaynakça
- Adaszek Ł, Jankowska M, Kalinowski M ve ark. (2013): The loop-mediated isothermal amplification assay for rapid diagnosis of Babesia canis canis infections in dogs. Pol J Vet Sci, 16, 131-3.
- Alhassan A, Govind Y, Tam NT ve ark. (2007): Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res, 100, 1165-1168.
- Alhassan A, Thekisoe OM, Yokoyama N ve ark. (2007): Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis. Vet Parasitol, 143, 155-160.
- Aonuma H, Suzuki M, Iseki H ve ark. (2008): Rapid identification of Plasmodium carrying mosquitoes using loop- mediated isothermal amplification. Biochem Biophys Res Commun, 376, 671-676.
- Bakheit MA, Torra D, Palomino LA ve ark. (2008): Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Vet Parasitol, 158, 11-22.
- Bilgic HB, Karagenc T, Shiels B ve ark. (2010): Evaluation of cytochrome b as a sensitive target for PCR based detection of T.annulata carrier animals. Vet Parasitol, 174, 341-347.
- Gill P, Ghaemi A (2008): Nucleic acid isothermal amplification Technologies-a review. Nucleos Nucleot Nucl, 27, 224-243.
- Guan G, Chauvin A, Luo J ve ark. (2008): The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China. Exp Parasitol, 120, 39-44.
- Ikadai H, Tanaka H, Shibahara N ve ark. (2004): Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J Clin Microbiol, 42, 2465-2469.
- Iseki H, Alhassan A, Ohta N ve ark. (2007): Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J Microbiol Methods, 71, 281-287.
- Kaneko H, Kawana T, Fukushima E ve ark. (2007): Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods, 70, 499-501.
- Karanis P, Thekisoe O, Kiouptsi K ve ark. (2007): Development and preliminary evaluation of a loop- mediated isothermal amplification procedure for sensitive detection of cryptosporidium oocysts in fecal and water samples. Appl Environ Microbiol, 73, 5660-5662
- Kuboki N, Inoue N, Sakurai T ve ark. (2003): Loop- mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol, 41, 5517-5524.
- Liu A, Guan G, Du P ve ark. (2012): Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina. Parasitol Int, 61, 658-63.
- Liu A, Guan G, Du P ve ark. (2012): Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences. Exp Parasitol, 131, 125-129.
- Liu Z, Hou J, Bakheit MA ve ark. (2008): Development of loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of ovine theileriosis in China. Parasitol Res, 103, 1407-1412.
- Mandal M, Banerjee PS, Kumar S ve ark. (2015): Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs. Vet Parasitol, 209, 50-55.
- Mori Y, Nagamine K, Tomita N ve ark. (2001): Detection of loop-mediated isothermal amplification reaction by turbidity formation. Biochem Biophys Res Commun, 289, 150-154. magnesium pyrophosphate
- Müller H, Aysul N, Liu Z ve ark. (2010): Development of a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Babesia canis infections. Transbound Emerg Dis, 57, 63-65.
- Nagamine K, Watanabe K, Ohtsuka K ve ark. (2001): Loop-mediated isothermal amplification reaction using a nondenatured template. Clin Chem, 47, 1742-1743.
- Notomi T, Okayama H, Masubuchi H ve ark. (2000): Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 15, 28(12): E63.
- Njiru ZK, Mikosza AS, Matovu E ve ark. (2008): African trypanosomiasis: Sensitive and rapid detection of the sub- genus amplification (LAMP) of parasite DNA. Int J Parasitol, 38, 589-599. loop-mediated isothermal and species distribution of caprine
- Parida M, Sannarangaiah S, Dash PK ve ark. (2008): Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol, 18, 407-421.
- Plutzer J, Karanis P (2009): Rapid identification of Giardia amplification (LAMP) from faecal and environmental samples and comparative findings by PCR and real-time PCR methods. Parasitol Res, 104, 1527-1533. isothermal
- Salih DA, Ali AM, Liu Z ve ark. (2012): Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi. Parasitol Res, 110, 533- 538.
- Salih DA, Liu Z, Bakheit MA ve ark. (2008): Development and evaluation of a loop-mediated isothermal amplification method for diagnosis of tropical theileriosis. Transbound Emerg Dis, 55, 238-243.
- Thekisoe OM, Bazie RS, Coronel-Servian AM ve ark. (2009): Stability of loop-mediated isothermal amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates. J Vet Med Sci, 71, 471-475.
- Thekisoe OM, Rambritch NE, Nakao R ve ark. (2010): Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes. Int J Parasitol, 40, 55-61.
- Tomita N, Mori Y, Kanda H ve ark. (2008): Loop- mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc, 3, 877-882.
- Xie J, Liu G, Tian Z ve ark. (2013): Development of loop- mediated isothermal amplification (LAMP) for detection of Theileria equi. Acta Trop, 127, 245-250. Geliş tarihi:28.09.2015 / Kabul tarihi: 13.10.2016
Öz
Son yıllarda polimeraz zincir reaksiyonuna (PZR) alternatif olarak termal siklus kullanmadan izotermal koş ullar altında hedef bölgelerin çoğaltılmasına olanak sağlayan LAMP (loop mediated isothermal amplification) gibi yeni teknikler geliştirilmiştir. LAMP, yüksek özgünlükte ve kısa zamanda çok sayıda hedef DNA kalıbının çoğaltılabildiği ve elde edilen ürünün kolay ve hızlı tespitine olanak veren bir yöntemdir. Bu çalışmada, Theileria annulata’nın merozoit yüzey antijeni (Mero1), 30 kDa major merozoit yüzey antijeni (Tams-1) ve sitokrom b genlerini çoğaltmak için özgül olarak tasarlanan primer çiftleri kullanılarak, hasta ve taşıyıcı sığırlarda T. annulata’nın tespitinde LAMP yönteminin özgüllük ve duyarlılığı değerlendirilmiştir. En yüksek duyarlılığa sahip olan primer çiftleri ile LAMP’ın sahadan toplanan örneklerde uygulanabilirliği araştırılmıştır. Sitokrom b genine özgü iki primer çiftinin (CYTOB1 ile CYTOB341) T. annulata’nın farklı izolatlarını özgül olarak çoğalttığı ve BL20 ve diğer türlere ait DNA’larda ise herhangi bir çoğalma olmadığı belirlenmiştir. CYTOB1 primerleri 2 fg’a kadar T. annulata Ankara / D7 DNA’sını tespit edebilmiş, bununla birlikte CYTOB341’in duyarlılığı CYTOB1’ye oranla 10 kat düşük bulunmuştur. Yapılan analizlerde, CYTOB1 LAMP’ının duyarlılığı F3/B3 PZR ile aynı bulunmuş, ancak CYTOB1 F3/B3 PZR'in duyarlılığının cytob1 PZR’ye oranla 10 kat daha az olduğu belirlenmiş tir. Ayrıca, LAMP ürünlerinin özgüllükleri restriksiyon enzimi ile keserek ve sekans analizleri ile doğrulanmıştır. Elde edilen sonuçlar, sitokrom b geni dışında hedef gen bölgelerine (Tams-1 ve Mero1) özgü tasarlanan primer çiftlerinin hiçbirinin T. annulata’nın farklı izolatlarını özgül ve duyarlı olarak tespit etmediğini göstermiştir. Sonuç olarak, CYTOB1 LAMP yönteminin T. annulata’nın saha şartlarında tespitinde kullanılabilirliğinin cytob1 PZR’ye oranla düş ük olduğu saptanmıştır
Anahtar Kelimeler
Kaynakça
- Adaszek Ł, Jankowska M, Kalinowski M ve ark. (2013): The loop-mediated isothermal amplification assay for rapid diagnosis of Babesia canis canis infections in dogs. Pol J Vet Sci, 16, 131-3.
- Alhassan A, Govind Y, Tam NT ve ark. (2007): Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res, 100, 1165-1168.
- Alhassan A, Thekisoe OM, Yokoyama N ve ark. (2007): Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis. Vet Parasitol, 143, 155-160.
- Aonuma H, Suzuki M, Iseki H ve ark. (2008): Rapid identification of Plasmodium carrying mosquitoes using loop- mediated isothermal amplification. Biochem Biophys Res Commun, 376, 671-676.
- Bakheit MA, Torra D, Palomino LA ve ark. (2008): Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Vet Parasitol, 158, 11-22.
- Bilgic HB, Karagenc T, Shiels B ve ark. (2010): Evaluation of cytochrome b as a sensitive target for PCR based detection of T.annulata carrier animals. Vet Parasitol, 174, 341-347.
- Gill P, Ghaemi A (2008): Nucleic acid isothermal amplification Technologies-a review. Nucleos Nucleot Nucl, 27, 224-243.
- Guan G, Chauvin A, Luo J ve ark. (2008): The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China. Exp Parasitol, 120, 39-44.
- Ikadai H, Tanaka H, Shibahara N ve ark. (2004): Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J Clin Microbiol, 42, 2465-2469.
- Iseki H, Alhassan A, Ohta N ve ark. (2007): Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J Microbiol Methods, 71, 281-287.
- Kaneko H, Kawana T, Fukushima E ve ark. (2007): Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods, 70, 499-501.
- Karanis P, Thekisoe O, Kiouptsi K ve ark. (2007): Development and preliminary evaluation of a loop- mediated isothermal amplification procedure for sensitive detection of cryptosporidium oocysts in fecal and water samples. Appl Environ Microbiol, 73, 5660-5662
- Kuboki N, Inoue N, Sakurai T ve ark. (2003): Loop- mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol, 41, 5517-5524.
- Liu A, Guan G, Du P ve ark. (2012): Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina. Parasitol Int, 61, 658-63.
- Liu A, Guan G, Du P ve ark. (2012): Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences. Exp Parasitol, 131, 125-129.
- Liu Z, Hou J, Bakheit MA ve ark. (2008): Development of loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of ovine theileriosis in China. Parasitol Res, 103, 1407-1412.
- Mandal M, Banerjee PS, Kumar S ve ark. (2015): Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs. Vet Parasitol, 209, 50-55.
- Mori Y, Nagamine K, Tomita N ve ark. (2001): Detection of loop-mediated isothermal amplification reaction by turbidity formation. Biochem Biophys Res Commun, 289, 150-154. magnesium pyrophosphate
- Müller H, Aysul N, Liu Z ve ark. (2010): Development of a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Babesia canis infections. Transbound Emerg Dis, 57, 63-65.
- Nagamine K, Watanabe K, Ohtsuka K ve ark. (2001): Loop-mediated isothermal amplification reaction using a nondenatured template. Clin Chem, 47, 1742-1743.
- Notomi T, Okayama H, Masubuchi H ve ark. (2000): Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 15, 28(12): E63.
- Njiru ZK, Mikosza AS, Matovu E ve ark. (2008): African trypanosomiasis: Sensitive and rapid detection of the sub- genus amplification (LAMP) of parasite DNA. Int J Parasitol, 38, 589-599. loop-mediated isothermal and species distribution of caprine
- Parida M, Sannarangaiah S, Dash PK ve ark. (2008): Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol, 18, 407-421.
- Plutzer J, Karanis P (2009): Rapid identification of Giardia amplification (LAMP) from faecal and environmental samples and comparative findings by PCR and real-time PCR methods. Parasitol Res, 104, 1527-1533. isothermal
- Salih DA, Ali AM, Liu Z ve ark. (2012): Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi. Parasitol Res, 110, 533- 538.
- Salih DA, Liu Z, Bakheit MA ve ark. (2008): Development and evaluation of a loop-mediated isothermal amplification method for diagnosis of tropical theileriosis. Transbound Emerg Dis, 55, 238-243.
- Thekisoe OM, Bazie RS, Coronel-Servian AM ve ark. (2009): Stability of loop-mediated isothermal amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates. J Vet Med Sci, 71, 471-475.
- Thekisoe OM, Rambritch NE, Nakao R ve ark. (2010): Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes. Int J Parasitol, 40, 55-61.
- Tomita N, Mori Y, Kanda H ve ark. (2008): Loop- mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc, 3, 877-882.
- Xie J, Liu G, Tian Z ve ark. (2013): Development of loop- mediated isothermal amplification (LAMP) for detection of Theileria equi. Acta Trop, 127, 245-250. Geliş tarihi:28.09.2015 / Kabul tarihi: 13.10.2016
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Veteriner Cerrahi |
| Diğer ID | JA44FK68UR |
| Bölüm | Araştırma Makalesi |
| Yazarlar | |
| Yayımlanma Tarihi | 1 Eylül 2017 |
| Yayımlandığı Sayı | Yıl 2017Cilt: 64 Sayı: 3 |