Deteksi Brucella abortus dari Sampel Darah-Utuh dengan Uji Polymerase Chain Reaction Tanpa Ekstraksi DNA

Abstract Brucellosis is a zoonotic disease that cause a significant economic losses for cattle industries worldwide. A rapid, precise and accurate diagnosis technique for diagnosis of brucellosis in all stages of the infection is definitely required. Blood-samples are widely used for PCR-based DNA analysis because they are easily collected, handled, and processed. Direct PCR analysis without DNA extraction has been attempted to reduce time and costs for routine analysis. This approach is promising but is still limited by the presence of PCR inhibitors that is naturally found in the blood samples. The objective of this study was to compare the effectivity of direct PCR technique with or without DNA extraction for detection of Brucella abortus in the blood samples. Three whole-blood samples from brucella infected dairy cattle and five whole-blood samples from beef cattle that having abortion were used as samples in this study. A pair of bcsp31 primers and IS711 primers were used for amplification of genus-specific and species-specific of Brucella. The results showed that amplicon in the position of 223 bp and 498 bp that are specific for B. abortus were detected from all of the samples that were analyzed on 1.5% agarose gels. Based on the result it could be concluded that direct PCR analyses without DNA extraction is a sensitive, specific, simple, rapid and inexpensive assay for detecting B. abortus in the whole blood samples for either dairy or beef cattle and therefore it could improve the existing surveillance and control programs for brucellosis. Keywords : brucellosis; direct PCR; PCR inhibitor; whole-blood sample; without DNA extraction Abstrak Brucellosis adalah penyakit zoonosis yang menyebabkan kerugian ekonomi yang signifikan bagi industri ternak di seluruh dunia. Teknik diagnosis yang cepat, tepat dan akurat yang dapat digunakan untuk diagnosis brucellosis pada semua tahap infeksi sangat diperlukan. Sampel darah banyak digunakan untuk analisis PCR berbasis DNA karena mudah untuk dikoleksi, ditangani, dan diproses. Metoda PCR langsung tanpa didahului dengan ekstraksi DNA dikembangkan dengan tujuan penghematan waktu dan beaya untuk analisa secara rutin. Tehnik ini sangat menjanjikan tetapi memiliki keterbatasan karena adanya senyawa penghambat PCR yang secara alami terkandung di dalam sampel darah . Tujuan dari penelitian ini adalah membandingkan efektifitas antara uji PCR secara langsung dengan ekstraksi dan tanpa ekstraksi DNA untuk deteksi Brucella abortus di dalam darah. Tiga ( 3 ) sampel darah-EDTA yang berasal dari sapi penderita brucellosis dan 5 sampel darah-EDTA dari sapi potong yang mengalami abortus digunakan sebagai sampel dalam penelitian ini. Pasangan primer bcsp31 dan primer IS711 untuk amplifikasi gen dan species specific digunakan dalam penelitian. Hasil menunjukkan bahwa amplikon/pita pada posisi 223 bp dan 498 bp yang spesifik untuk Brucella abortus terdeteksi dari semua sampel yang dianalisa dengan gel agarosa 1,5%. Berdasarkan hasil penelitian dapat disimpulkan bahwa uji PCR secara langsung tanpa didahului dengan ekstraksi DNA merupakan tehnik yang sensitif, spesifik, sederhana, cepat dan murah untuk deteksi B. abortus di dalam sampel darah baik sapi perah maupun sapi potong dan oleh karena itu diharapkan dapat digunakan untuk memperbaiki program kontrol dan survailance yang telah ada untuk brucellosis. Kata kunci : brucellosis; PCR langsung; penghambat PCR; sampel darah-utuh; tanpa ekstraksi DNA

Download Full-text

Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods

Experimental Parasitology ◽

10.1016/j.exppara.2014.08.006 ◽

2014 ◽

Vol 146 ◽

pp. 20-24 ◽

Cited By ~ 7

Author(s):

J. Dunlop ◽

C.K. Thompson ◽

S.S. Godfrey ◽

R.C.A. Thompson

Keyword(s):

Dna Extraction ◽

Whole Blood ◽

Extraction Methods ◽

Sensitivity Testing ◽

Blood Samples ◽

Whole Blood Samples ◽

Automated Dna Extraction ◽

Dna Extraction Methods

Download Full-text

Chemiluminescence Analysis for HBV-DNA Hybridization Detection with Magnetic Nanoparticles Based DNA Extraction from Positive Whole Blood Samples

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2013.1478 ◽

2013 ◽

Vol 9 (2) ◽

pp. 267-273 ◽

Cited By ~ 18

Author(s):

Nongyue He ◽

Fang Wang ◽

Chao Ma ◽

Chuanyan Li ◽

Xin Zeng ◽

...

Keyword(s):

Magnetic Nanoparticles ◽

Dna Extraction ◽

Whole Blood ◽

Dna Hybridization ◽

Hbv Dna ◽

Blood Samples ◽

Chemiluminescence Analysis ◽

Whole Blood Samples ◽

Hybridization Detection

Download Full-text

Comparison of genomic DNA extraction techniques from whole blood samples: a time, cost and quality evaluation study

Molecular Biology Reports ◽

10.1007/s11033-011-1408-8 ◽

2012 ◽

Vol 39 (5) ◽

pp. 5961-5966 ◽

Cited By ~ 26

Author(s):

Diego Chacon-Cortes ◽

Larisa M. Haupt ◽

Rod A. Lea ◽

Lyn R. Griffiths

Keyword(s):

Dna Extraction ◽

Whole Blood ◽

Genomic Dna ◽

Quality Evaluation ◽

Evaluation Study ◽

Time Cost ◽

Extraction Techniques ◽

Blood Samples ◽

Genomic Dna Extraction ◽

Whole Blood Samples

Download Full-text

Molecular Identification of Brucella abortus Collected from Whole Blood Samples of Seronegative Dairy Cattle with Reproductive Disorders in Central Java, Indonesia

Pakistan Veterinary Journal ◽

10.29261/pakvetj/2019.010 ◽

2019 ◽

Vol 39 (03) ◽

pp. 455-458

Author(s):

Hastari Wuryastuty

Keyword(s):

Dairy Cattle ◽

Molecular Identification ◽

Brucella Abortus ◽

Whole Blood ◽

Reproductive Disorders ◽

Blood Samples ◽

Central Java ◽

Whole Blood Samples

Download Full-text

Comparison of commercial DNA extraction kits for the detection of bacterial genomic DNA from whole-blood samples using a broad-range PCR

Critical Care ◽

10.1186/cc8069 ◽

2009 ◽

Vol 13 (Suppl 4) ◽

pp. P13

Author(s):

B Krulova ◽

E Nemcova ◽

B Zaloudikova ◽

P Nemec ◽

T Freiberger

Keyword(s):

Dna Extraction ◽

Whole Blood ◽

Genomic Dna ◽

Blood Samples ◽

Whole Blood Samples

Download Full-text

A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.758564 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xinggui Yang ◽

Yue Wang ◽

Ying Liu ◽

Junfei Huang ◽

Qinqin Tan ◽

...

Keyword(s):

Brucella Abortus ◽

Whole Blood ◽

Isothermal Amplification ◽

Polymer Nanoparticles ◽

Bovine Brucellosis ◽

Isolation And Identification ◽

Blood Samples ◽

Diagnostic Strategies ◽

Loop Mediated Isothermal Amplification ◽

Whole Blood Samples

Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot meet the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of B. abortus, but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive, and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of B. abortus, which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific B. abortus-LAMP primers targeting the BruAb2_0168 gene was designed by the online LAMP primer design tool. The B. abortus-LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for B. abortus-LAMP-LFIA were determined to be 65°C and 50 min, respectively. The B. abortus-LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of B. abortus. Meanwhile, the detection specificity was 100%, and there was no cross-reactivity for other Brucella members and non-Brucella strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min), and LFIA detection (2–5 min), can be completed in approximately 85 min. Thus, the B. abortus-LAMP-LFIA assay developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.

Download Full-text

Sensitivity and specificity levels of two rapid assays for antibodies to Anaplasma spp. in dogs

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638717745932 ◽

2017 ◽

Vol 30 (2) ◽

pp. 290-293 ◽

Cited By ~ 3

Author(s):

Jiayou Liu ◽

Matthew Eberts ◽

Hannah Bewsey ◽

Thomas P. O’Connor ◽

Ramaswamy Chandrashekar ◽

...

Keyword(s):

United States ◽

Whole Blood ◽

Rapid Test ◽

Blood Samples ◽

Rapid Tests ◽

Tick Borne Disease ◽

Whole Blood Samples ◽

Species Specific ◽

Tick Vectors ◽

Sample Set

Canine anaplasmosis is a tick-borne disease of dogs that results following infection with Anaplasma phagocytophilum or Anaplasma platys. The SNAP 4Dx Plus test (IDEXX Laboratories) and the VetScan Canine Anaplasma Rapid test (Abaxis) are commercial in-house rapid tests for the detection of antibody to these 2 antigenically related Anaplasma species. We evaluated 2 tests using serum and whole blood samples obtained from reference laboratories and veterinary hospitals. Samples were obtained from regions of the country known to be habitats of the primary tick vectors. The A. phagocytophilum sample set comprised 236 dog sera from the northeastern and midwestern United States; the A. platys sample set comprised 179 sera from dogs living in the southwestern United States. An indirect immunofluorescent antibody (IFA) test and an A. platys species-specific ELISA were used as reference assays for the A. phagocytophilum and A. platys samples, respectively. The SNAP test demonstrated significantly higher sensitivity (84.7% for A. phagocytophilum and 83.1% for A. platys), compared to the VetScan test (39.0% for A. phagocytophilum and 57.6% for A. platys). The specificity of the SNAP test (95.8% for A. phagocytophilum and 99.2% for A. platys) was significantly greater than the VetScan test (85.6% for A. phagocytophilum and 82.5% for A. platys). In a separate clinic study, conducted within an A. phagocytophilum–endemic state (Minnesota) using 154 whole blood samples from client-owned dogs, the VetScan test was negative for 22 of 39 SNAP and IFA seropositive samples.

Download Full-text