scholarly journals Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

2021 ◽  
Vol 14 (1) ◽  
pp. 144-154
Author(s):  
Nour H. Abdel-Hamid ◽  
Eman I. M. Beleta ◽  
Mohamed A. Kelany ◽  
Rania I. Ismail ◽  
Nadia A. Shalaby ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Roc Curve ◽  
Diagnostic Performance ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Serum Samples ◽  
Polymerase Chain ◽  
Pcr Targeting ◽  
Pcr Techniques

Background and Aim: Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real- Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera. Materials and Methods: One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard. Results: TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%. Conclusion: A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

scholarly journals Use of immunodot blot and multiplex reverse transcriptase-polymerase chain reaction in dengue virus detection in macerates of Aedes aegypti larvae

2012 ◽  
Vol 3 (1) ◽  
pp. 13
Author(s):  
Aline T.A. Chagas ◽  
Michelle D. Oliveira ◽  
Jose M.S. Mezencio ◽  
Eduardo A.M. Silva ◽  
Leandro L. Oliveira ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Aedes Aegypti ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Epidemiological Surveillance ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Denv Serotypes ◽  
Polymerase Chain ◽  
Pcr Techniques

The <em>Dengue virus</em> is the main arbovirus that affects man in terms of morbidity and mortality. The detection of the virus is very important for epidemiological surveillance, so here we propose to standardize and compare the immunodot blot (IDB) and multiplex reverse transcriptase-polymerase chain reaction (M-RT-PCR) techniques to detect and characterize the dengue virus (DENV) serotypes in samples of <em>Aedes aegypti</em> larvae. Thus, the IDB and M-RT-PCR techniques were standardized using macerated samples of larvae collected in nature. The use of monoclonal antibodies in IDB has not shown great results, but DENV detection through this method was possible using polyclonal antibodies. The distinction of serotypes 1, 2 and 3 was carried out by M-RT-PCR.

scholarly journals Evaluation of the Reverse Transcription-Polymerase Chain Reaction/Probe Test of Serum Samples and Immunohistochemistry of Skin Sections for Detection of Acute Bovine Viral Diarrhea Infections

2002 ◽  
Vol 14 (4) ◽  
pp. 303-307 ◽  
Author(s):  
Julia F. Ridpath ◽  
Sharon K. Hietala ◽  
Steve Sorden ◽  
John D. Neill
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Amplification ◽  
Clinical Signs ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Serum Samples ◽  
Viral Diarrhea ◽  
Persistent Infections ◽  
Polymerase Chain

Bovine viral diarrhea viruses (BVDV) cause both acute and persistent infections. While diagnostic tests have been designed to detect animals persistently infected (PI) with BVDV, the reliability of these tests in detecting acute BVDV infections is not known. It is also possible that acute BVDV infections may be confused with persistent infections in surveys for PI animals. In this study, 2 tests presently in use in diagnostic laboratories to test for PI animals, polymerase chain reaction amplification followed by probe hybridization (RT-PCR/probe) of serum samples and immunohistochemical detection of viral antigen in skin biopsies (IHC), were evaluated for their ability to detect acute BVDV infections. Sixteen colostrum-deprived, BVDV-free, and BVDV-antibody-free calves were infected with 6 different BVDV strains. Clinical signs, seroconversion, and virus isolation indicated that inoculated animals did replicate virus. Virus could be detected in 19% (3/16) of acutely infected animals by the RT-PCR/probe technique. No acutely infected animals were positive by IHC.

scholarly journals Clinical Usefulness of Nested Reverse-Transcription Polymerase Chain Reaction for the Diagnosis of Severe Fever with Thrombocytopenia Syndrome

2021 ◽  
Author(s):  
Choon Mee Kim ◽  
Dong-Min Kim ◽  
Na-Ra Yun
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Immunofluorescence Assay ◽  
Clinical Usefulness ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Pcr Targeting ◽  
Severe Fever

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) is an emerging tick-borne infectious disease. Few studies have assessed the clinical usefulness of nested reverse-transcription polymerase chain reaction (RT-PCR) for diagnosing SFTS. We performed conventional RT-PCR targeting the M segment, nested RT-PCR targeting M and S segments, and real-time RT-PCR targeting the S segment of SFTSV for four patients with suspected SFTS. Although conventional RT-PCR results for the first two patients were negative at admission, nested RT-PCR using the S or M targets was positive for the same samples. Likewise, in the other two patients, initial samples were confirmed positive in all three tests, but follow-up testing demonstrated negative conventional RT-PCR and positive nested RT-PCR results. Thus, delayed testing using conventional RT-PCR or real-time RT-PCR in symptomatic patients with SFTS may result in missed diagnoses, and compared with these methods, nested RT-PCR may increase the window for obtaining positive SFTSV PCR results. Meanwhile, the indirect immunofluorescence assay showed seroconversion to SFTSV antibodies in all four patients. Nested RT-PCR for SFTSV M and S segments could help diagnose SFTS in patients testing negative by conventional RT-PCR.

scholarly journals Analysis of the demand for detection of SARSCoV-2 in a health area of Spain

2000 ◽  
Vol 33 (6) ◽  
pp. 422-429
Author(s):  
Jesús García-Cruces ◽  
Raúl López Izquierdo ◽  
Marta Domínguez-Gil ◽  
Luis López-Urrutia ◽  
Mónica de Frutos ◽  
...  
Keyword(s):  
Primary Care ◽  
Polymerase Chain Reaction ◽  
Positive Result ◽  
Diagnostic Performance ◽  
Descriptive Analysis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Health Area ◽  
Reference Hospital ◽  
Polymerase Chain

Introduction. Since the discovery of the SARS-CoV-2 virus, the polymerase chain reaction technique (RT-PCR) has become the fundamental method for diagnosing the disease in its acute phase. The objective is to describe the demand-based series of RT-PCR determinations received at a Microbiology Service at a third-level reference hospital for a health area for three months spanning from the onset of the epidemic by SARS-CoV-2. Methods. A retrospective analysis of the total of the RT-PCR requested in the Microbiology Service analyzed from 02/25/2020 to 05/26/2020 (90 days) has been carried out. They have been grouped by epidemiological weeks and by the petitioner service. A descriptive analysis was carried out by age, gender and number of requests for each patient. In the tests carried out, a confidence level of 95% (p <0.05) was considered significant. Results. A total of 27,106 requests was received corresponding to 22,037 patients. Median age 53.7 (RIC 40.9-71.7) years, women: 61.3%. Proportion of patients with any positive RT-PCR: 14%. Of the total requests for RT-PCR, positive 3,710. Week 13 had the highest diagnosis performance (39.0%). The primary care has been the service thar has made the most requests (15,953). Patients with 3 or more RT-PCR: 565, of them, 19 patients had a positive result after previously having a negative one. Conclusions. Requests have been increasing depending on the evolution of the epidemic. The RT-PCR has a high diagnostic performance in the phases of highest contagiousness and / or transmissibility of the virus.

scholarly journals DETEKSI VIRUS JAPANESE ENCEPHALITIS PADA MANUSIA DAN VEKTOR DI KABUPATEN TULUNGAGUNG, JAWA TIMUR

2020 ◽  
Vol 12 (1) ◽  
pp. 33-40
Author(s):  
Nova Pramestuti ◽  
Tri Wijayanti ◽  
Dyah Widiastuti ◽  
Tri Isnani
Keyword(s):  
Polymerase Chain Reaction ◽  
Human Serum ◽  
Japanese Encephalitis ◽  
Immunosorbent Assay ◽  
Culex Quinquefasciatus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Serum Samples ◽  
Polymerase Chain ◽  
Anopheles Vagus

Japanese encephalitis is a zoonotic disease transmitted by mosquitoes with pigs as the main reservoir. A small percentage of infected people experience acute encephalitis syndrome, with one in four cases fatal. Recently, the existence of a growing pig population has the potential to increase the risk of JE transmission in Tulung Agung, East Java, Indonesia. The purpose of this study was to detect JE infection in humans and mosquitoes in Tulungagung Regency. A cross-sectional design was conducted from March to October 2016. Human blood screening was carried out in six hospitals in Tulungagung and the mosquitos survey was carried out using human landing collection, resting collection, and light trap around the pig farms in Kaliwungu District. Detection of JE infection was carried out by indirect immuno-linked immunosorbent assay (ELISA) testing of IgM/IgG in human serum samples and Polymerase Chain Reaction (RT-PCR). Transcription reaction in mosquitoes. The ELISA test showed that one of 19 human serum samples was confirmed positive with JE specific IgG. The result of the mosquito survey showed that Anopheles vagus was predominantly collected in Kaliwungu village, while Culex quinquefasciatus was was predominantly species collected in Pulosari Village. The analyses using molecular assays showed that all captured mosquitoes were negative Javanese encephalitis virus (JEV).  Abstrak Japanese Encephalitis (JE) merupakan penyakit zoonotik yang ditularkan oleh nyamuk dengan reservoir utama babi. Sebagian kecil orang yang terinfeksi mengalami radang otak (ensefalitis), sekitar satu dari empat kasus berakibat fatal. Peningkatan populasi ternak babi di Kabupaten Tulungagung berpotensi menyebarkan virus JE. Tujuan penelitian untuk mendeteksi infeksi JE pada manusia dan nyamuk di Kabupaten Tulungagung. Penelitian dilakukan pada Bulan Maret-Oktober tahun 2016 dengan desain studi potong lintang. Survey darah manusia dilakukan pada enam rumah sakit di Kabupaten Tulungagung. Survei nyamuk dilakukan satu kali dengan metode umpan badan manusia dan perangkap cahaya, serta penangkapan nyamuk istirahat di sekitar peternakan babi di Kecamatan Kaliwungu. Deteksi infeksi JE dilakukan dengan pemeriksaan Indirect Enzyme-Linked Immunosorbent Assay (ELISA) IgM/IgG pada sampel serum manusia dan Reverse Transcription Polymerase Chain Reaction (RT-PCR) pada nyamuk. Hasil pemeriksaan laboratorium terhadap antibodi IgM/IgG JE menunjukkan satu kasus positif IgG JE dari 19 sampel serum manusia yang diperiksa. Spesies nyamuk yang tertangkap di Desa Kaliwungu didominasi Anopheles vagus, sedangkan di Desa Pulosari didominasi Culex quinquefasciatus. Hasil pemeriksaan RT-PCR terhadap semua sampel nyamuk yang tertangkap menunjukkan negatif virus JE. Satu pasien ditemukan positif antibodi IgG Japanese encephalitis di Kabupaten Tulungagung.

scholarly journals Reverse transcription and polymerase chain reaction: principles and applications in dentistry

2004 ◽  
Vol 12 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Carlos Ferreira dos Santos ◽  
Vivien Thiemy Sakai ◽  
Maria Aparecida de Andrade Moreira Machado ◽  
Daniela Nicole Schippers ◽  
Andrew Seth Greene
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
High Specificity ◽  
Rt Pcr ◽  
Chain Reaction ◽  
New Techniques ◽  
Treatment And Prevention ◽  
Nucleic Acid Analysis ◽  
Polymerase Chain ◽  
Pcr Techniques

Various molecular biology techniques have become available in the last few years. One of the most revolutionary of these techniques regarding nucleic acid analysis is the polymerase chain reaction (PCR), which was first described in 1985. This method relies on the exponential amplification of specific DNA fragments, resulting in millions of copies that can serve as templates for different kinds of analyses. PCR can be preceded by a reverse transcription (RT) reaction in order to produce cDNA from RNA (RT-PCR). RT-PCR provides the possibility to assess gene transcription in cells or tissues. PCR and RT-PCR techniques have been instrumental in dental research, and show potential to be used for diagnosis as well as for treatment and prevention of many diseases (dental caries, periodontal disease, endodontic infections and oral cancer). Compared to other traditional methodologies, PCR and RT-PCR show many advantages including high specificity, sensitivity, and speed. Since PCR and RT-PCR are relatively new techniques and are not available to most students and professionals involved with dentistry, the aim of this work is to present the details of these techniques as well as dental literature reports in which they were used.

scholarly journals Usefulness of the extraction-free RT-PCR methods for the SARS-CoV-2 diagnostics: An Italian experience

2021 ◽  
Author(s):  
Alexander Domnich ◽  
Vanessa De Pace ◽  
Beatrice M. Pennati ◽  
Patrizia Caligiuri ◽  
Serena Varesano ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Gold Standard ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Perfect Agreement ◽  
Valuable Alternative ◽  
Italian Experience ◽  
Heat Treated ◽  
Polymerase Chain ◽  
Pcr Techniques

Abstract The extraction-based real-time reverse transcription polymerase chain reaction (RT-PCR) is currently the “gold standard” for the SARS-CoV-2 diagnostics. However, some extraction-free RT-PCR techniques have been recently developed. In this study we compared the performance of heated and unheated extraction-free methods with the traditional extraction-based SARS-CoV-2 RT-PCR. The unheated extraction-free showed a perfect agreement with the standard extraction-based RT-PCR. By contrast, the heat-treated technique was associated with an 8.2% false negativity rate. The unheated extraction-free RT-PCR for the SARS-CoV-2 molecular diagnostics is a valuable alternative to the traditional extraction-based methods and may accelerate turnaround times by about two hours.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Polymerase chain reaction amplification and typing of rotavirus in environmental samples

1995 ◽  
Vol 31 (5-6) ◽  
pp. 371-374 ◽  
Author(s):  
R. Gajardo ◽  
R. M. Pintó ◽  
A. Bosch
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Polymerase Chain Reaction Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Group A ◽  
Reaction Amplification ◽  
Stool Specimens ◽  
Polymerase Chain

A reverse transcription polymerase chain reaction (RT-PCR) assay is described that has been developed for the detection and serotyping of group A rotavirus in stool specimens and concentrated and non-concentrated sewage specimens.

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