scholarly journals Molecular Detection of Mycoplasma haemofelis and `Candidatus Mycoplasma haemominutum' Infection in Cats by Direct PCR Using Whole Blood without DNA Extraction

2008 ◽  
Vol 70 (10) ◽  
pp. 1095-1099 ◽  
Author(s):  
Masashi WATANABE ◽  
Masaharu HISASUE ◽  
Takehisa SOUMA ◽  
Shuichi OHSHIRO ◽  
Takatsugu YAMADA ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Whole Blood ◽  
Molecular Detection ◽  
Direct Pcr

scholarly journals Direct PCR from whole blood, without DNA extraction

1990 ◽  
Vol 18 (19) ◽  
pp. 5908-5909 ◽  
Author(s):  
B. Mercier ◽  
C. Gaucher ◽  
O. Feugeas ◽  
C. Mazurier
Keyword(s):  
Dna Extraction ◽  
Whole Blood ◽  
Direct Pcr

scholarly journals Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
R. R. Alyethodi ◽  
U. Singh ◽  
S. Kumar ◽  
R. Alex ◽  
G. S. Sengar ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Whole Blood ◽  
Cold Chain ◽  
Dna Testing ◽  
Direct Pcr ◽  
Cattle Industry ◽  
Vertebral Malformation ◽  
Arms Pcr ◽  
Optimized Protocol ◽  
Field Samples

Abstract Background DNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples. Results The present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards. Conclusions The present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.

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

2020 ◽  
Vol 38 (3) ◽  
pp. 222
Author(s):  
David Ardiyanto ◽  
Hastari Wuryastuty ◽  
Raden Wasito
Keyword(s):  
Beef Cattle ◽  
Dna Extraction ◽  
Brucella Abortus ◽  
Whole Blood ◽  
Economic Losses ◽  
Direct Pcr ◽  
Blood Samples ◽  
Pcr Inhibitor ◽  
Whole Blood Samples ◽  
Species Specific

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

scholarly journals Diagnosis of Bovine Theileriosis by Direct PCR and Electrophoresis from Whole Blood Without DNA Extraction

2003 ◽  
Vol 47 (2) ◽  
pp. 127-132 ◽  
Author(s):  
Seong Ho Kang ◽  
Sangmin Jang ◽  
Joon-Seok Chae ◽  
Yongseong Kim
Keyword(s):  
Dna Extraction ◽  
Whole Blood ◽  
Direct Pcr ◽  
Bovine Theileriosis

scholarly journals Detection of Bacteria in Environmental Samples by Direct PCR Without DNA Extraction

BioTechniques ◽  
2001 ◽  
Vol 31 (3) ◽  
pp. 598-607 ◽  
Author(s):  
Kimberly A. Fode-Vaughan ◽  
Charles F. Wimpee ◽  
Charles C. Remsen ◽  
Mary Lynne Perille Collins
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Direct Pcr

scholarly journals Molecular Detection of Sepsis-Causing Pathogens Directly From Whole Blood Specimens

2015 ◽  
Vol 2 (suppl_1) ◽  
Author(s):  
Alexandra Barr ◽  
Edward Adams ◽  
Lisa-Jo Clarizia ◽  
Ruth Bauer ◽  
Stephen Springer ◽  
...  
Keyword(s):  
Whole Blood ◽  
Molecular Detection ◽  
Blood Specimens

scholarly journals Molecular detection and occurrence of equine theileriosis in Arabian horses in Al-Najaf province/Iraq

2020 ◽  
Vol 57 (3) ◽  
pp. e166996
Author(s):  
Hayder Mohammad Al-Rammahi ◽  
Abdulameer Abed Hatem ◽  
Asaad Chasib Al-Atabi
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Molecular Detection ◽  
Molecular Technique ◽  
Chain Reaction ◽  
Blood Samples ◽  
Equine Piroplasmosis ◽  
Polymerase Chain

This study was designed to detect equine piroplasmosis using the molecular technique in Al-Najaf province during the season that showed an increment in tick activities. Blood samples were collected from 110 horses with more than two signs of piroplasmosis. After DNA extraction, the product was examined by a polymerase chain reaction to amplify 18SrRNA. The results showed that the overall percentage of equine theileriosis was 38.18%. According to gender, the percentage of infection was 43.48% and 29.27% in females and males, respectively. Significant variations appeared between infected horses according to age, and the percentage of infection was 50% and 35.22% in less than 2 years and more than 2 years age, respectively. Moreover, the percentage of infection was 62.5% and 19.35% in animals with and without acariasis, respectively. Significant variations were also seen in equine theileriosis according to geographical areas, and the higher percentage was reported in Hera district (60.87%), while the lowest percentage was in the center of Al-Najaf (21.43%). This difference may be due to different distribution of vector of disease (tick), which may be the availability of the suitable weather that helped in the multiplication of the intermediate vectors. In conclusion, this study proved the variations in the occurrences of equine piroplasmosis according to gender, age, and geographical areas.

scholarly journals A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta

2013 ◽  
Vol 58 (4) ◽  
pp. 350-355 ◽  
Author(s):  
C. Biswas ◽  
P. Dey ◽  
S. Satpathy
Keyword(s):  
Dna Extraction ◽  
Rapid Detection ◽  
Direct Pcr
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