scholarly journals Real-Time Polymerase Chain Reaction Approach for Quantitation of Ruminant-Specific DNA to Indicate a Correlation Between DNA Amount and Meat and Bone Meal Heat Treatments

2005 ◽  
Vol 88 (5) ◽  
pp. 1399-1403 ◽  
Author(s):  
Barbara Chiappini ◽  
Gianfranco Brambilla ◽  
Umberto Agrimi ◽  
Gabriele Vaccari ◽  
Henk J M Aarts ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
False Negative ◽  
The United States ◽  
Spongiform Encephalopathy ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Dna Amount ◽  
Polymerase Chain

Abstract The use of ruminant-derived proteins in ruminant feeds has been banned in both the European Union and the United States to prevent further spread of bovine spongiform encephalopathy. Enforcement of these regulations relies on the ability to identify the presence of prohibited proteins in feed. We developed a quantitative real-time polymerase chain reaction assay for the quantification of ruminant-specific DNA as index of protein content. The assay is based on the amplification of a 117 base pair mitochondrial 16S rRNA DNA gene fragment and an internal positive control (IPC). The use of an IPC permits compensation for differences in DNA extraction efficiency and avoids the occurrence of false-negative results. We demonstrated a decrease in target DNA amount with a difference of 2 logs between meat and bone meal (MBM) treated at 133° and 145°C. Such a difference indicates that bias could occur when DNA-based methods are used for quantitation purposes. Risk management could benefit from future efforts concerning validation of the method for MBM detection in feedstuff and safety evaluation of the use of animal-derived proteins in animal nutrition.

scholarly journals Collaborative Trial for Validation of a Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay for Detection of Central Nervous System Tissues as Bovine Spongiform Encephalopathy RiskMaterial: Part 1

2006 ◽  
Vol 89 (5) ◽  
pp. 1335-1340
Author(s):  
Amir Abdulmawjood ◽  
Holger Schnenbrcher ◽  
Michael BÜlte
Keyword(s):  
Central Nervous System ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Products ◽  
Spongiform Encephalopathy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Heat Treated ◽  
Polymerase Chain

Abstract A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120C) and the medium heat-treated samples (sausages cooked at 80C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

scholarly journals Perspective of Arguments in Public Health

2021 ◽  
Vol 9 (1) ◽  
pp. 44-45
Author(s):  
Dinesh Kumar
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Respiratory Symptoms ◽  
False Negative ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Report ◽  
Polymerase Chain ◽  
Time Of Admission ◽  
Pcr Test

Recently, an argument was put forth because a symptomatic and positive patient for CoVID-19 turned tested negative after 7 days, so discharged from the hospital. Both at the time of admission and discharge real-time reverse transcriptase Polymerase Chain Reaction (RT-PCR) was done for testing of CoVID-19. Immediately, patient again developed respiratory symptoms and was admitted to hospital again. Amidst of current CoVID-19 pandemic, a question was asked “What is the specificity of the Real Time-Polymerase Chain Reaction (RT-PCR) test for COVID-19?” with an assumption that what if at the time of discharge the disease is present in patient but test turned out to be negative? In response to that a counter statement was posed that “It is the sensitivity that should be asked rather than specificity”. It was based on the implication of primary question that was implying false negative report of the RT-PCR. It means, since patient was discharged with negative result that could be false negative.

scholarly journals Reducing False Negative PCR Test for COVID-19

2020 ◽  
Vol 9 (3) ◽  
pp. 408-410
Author(s):  
Fatemeh Bahreini ◽  
Rezvan Najafi ◽  
Razieh Amini ◽  
Salman Khazaei ◽  
Saeid Bashirian
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
False Negative ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Creative Commons ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Pcr Test

As the SARS-CoV-2 (COVID-19) pandemic spreads rapidly, there is need for a diagnostic test with high accuracy to detect infected individuals especially those without symptoms. Real-time polymerase chain reaction (RT-PCR) is a common molecular test for diagnosing SARS-CoV-2. If some factors are not taken into consideration when performing this test, it can have a relatively large number of false negative results. In this article, we discuss important considerations that could lead to false negative test reduction. Key words: • SARS-CoV-2 • COVID-19 • Real time polymerase chain reaction • RT-PCR test • Diagnosis • False negatives • Genetics • Emerging disease   Copyright © 2020 Bahreini et al. Published by Global Health and Education Projects, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0)which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in this journal, is properly cited.

scholarly journals Real-Time Polymerase chain reaction trends in COVID-19 patients

2020 ◽  
Vol 37 (1) ◽  
Author(s):  
Dr Sana Abbas ◽  
Aisha Rafique ◽  
Dr Beenish Abbas ◽  
Dr Rashid Iqbal
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Tertiary Care ◽  
False Negative ◽  
Polymerase Chain Reaction Test ◽  
Upper Respiratory Tract ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Asymptomatic Patients ◽  
Polymerase Chain

Objective: To assess trends of real-time Polymerase Chain Reaction test in Coronavirus infected Patients. Methods: This cross-sectional analytical study was conducted at Tertiary Care Institute, Rawalpindi from March 2020 to June 2020. All patients confirmed COVID positive by real-time Polymerase Chain Reaction (PCR) with recent travel history, close contact with known diagnosed patients and had symptoms of fever or upper respiratory tract with body aches. Nasopharyngeal swabs were taken and results generated within 48 hours. Positive PCR was admission criteria follow up was carried out at 7th and 8th day, with negative PCR were discharged. However, those who had persistent positive PCR on the 8th day were tested again on 11th and 12th day. Those with persistent positive results beyond 12th day were shifted to specialized quarantine centres. Results: A total of three hundred and ninety-two patients with mild to moderate illness, PCR positive for COVID 19 were included study with age range 9 - 45 and mean 33.22±7.98 years. A total of 8 (2%) patients were females and 384(98%) males. The duration of the negative test result was Mean ± Std. Deviation 9.05±2.00 with 7 – 8 days 152(38.8%)in and 11 – 12 days in 160(40.8%). PCR results on Day 7 and 8 were negative in 144(36.7%) patients whereas positive in 248(63.3%). PCR results on Day 11 and 12 were negative in 312(79.6%) patients whereas positive in 80 (20.4%). Conclusion: To conclude Real-Time Polymerase Chain Reaction (rT-PCR) inclines to give false negative results additionally can stay positive in asymptomatic patients for moderately longer-term. Hence decision to discharge ought to be intricately adjusted between RT-PCR, clinical judgement, radiological examinations, and biochemical assays. doi: https://doi.org/10.12669/pjms.37.1.3000 How to cite this:Abbas S, Rafique A, Abbas B, Iqbal R. Real-Time Polymerase chain reaction trends in COVID-19 patients. Pak J Med Sci. 2021;37(1):180-184. doi: https://doi.org/10.12669/pjms.37.1.3000 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

scholarly journals A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

2006 ◽  
Vol 96 (11) ◽  
pp. 1255-1262 ◽  
Author(s):  
C. Zijlstra ◽  
R. A. Van Hoof
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Simultaneous Detection ◽  
Chain Reaction ◽  
Meloidogyne Chitwoodi ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.

scholarly journals A Competitive Polymerase Chain Reaction–Based Approach for the Identification and Semiquantification of Mitochondrial DNA in Differently Heat-Treated Bovine Meat and Bone Meal

2003 ◽  
Vol 66 (1) ◽  
pp. 103-109 ◽  
Author(s):  
DOMENICO FREZZA ◽  
MARCO FAVARO ◽  
GABRIELE VACCARI ◽  
CHRISTOPH von-HOLST ◽  
VINCENZO GIAMBRA ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Polymerase Chain Reaction ◽  
Mitochondrial Dna ◽  
Amplification Efficiency ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Animal Feeds ◽  
Heat Treated ◽  
Polymerase Chain

The risk of bovine spongiform encephalopathy propagation was drastically reduced after the European Union (EU) Health Authorities adopted restrictions involving a ban on animal-derived proteins in the diet of farm animals. Currently, the EU's officially recommended method for controlling meat and bone meal (MBM) in animal feed is the microscopic method, which involves the identification of bone fragments on the basis of their morphological characteristics. Recently, we demonstrated that a polymerase chain reaction (PCR)–based assay can be used for the detection of taxon-specific DNA in MBM and animal feeds. To ensure the safe rendering of animal by-products, the EU Council requires that this material be treated at 133°C at 300 kPa for 20 min. Here we investigate the relationship between DNA degradation, PCR amplification, and MBM heat treatment. With a competitive PCR-based approach, we compare the amplification efficiency of bovine mitochondrial DNA target sequences of different lengths in several heat-treated MBM samples. For our method, a synthetic competitive DNA is used as an internal control for both DNA extraction and PCR reaction. A correlation between an increase in treatment temperature and a reduction in the size of the target sequences suitable for amplification was observed, suggesting progressive DNA fragmentation due to the temperature. We show that short amplicons (147 bp) can be used to detect the presence of bovine mtDNA in MBM samples treated according to the current European regulations. The use of such a competitive approach to compare amplification efficiency levels of targets of different lengths might represent a useful tool for the determination of both the amount of MBM in animal feeds and its proper heat treatment.

First Documented Cases of Canine Neuroangiostrongyliasis Due to Angiostrongylus cantonensis in Hawaii

2020 ◽  
Vol 57 (1) ◽  
pp. 42-46
Author(s):  
Jenee Odani ◽  
Erika Sox ◽  
Will Coleman ◽  
Rajesh Jha ◽  
Richard Malik
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Complete Recovery ◽  
The United States ◽  
Angiostrongylus Cantonensis ◽  
Chain Reaction ◽  
Polymerase Chain Reaction Testing ◽  
Computed Tomography Scans ◽  
Polymerase Chain ◽  
Withdrawal Reflexes

ABSTRACT Two young dogs domiciled in Honolulu, Hawaii, were presented in November and December 2018 (respectively) for spinal hyperesthesia, hindlimb weakness, and proprioceptive ataxia. Both dogs had neurologic findings referable to spinal cord disease. Both dogs had a combination of lower motor neuron signs (reduced muscle mass, decreased withdrawal reflexes, low tail carriage) and long tract signs (conscious proprioceptive deficits, crossed extensor response, increased myotatic reflexes). Peripheral eosinophilia was present in the second case, but hematology and serum biochemistries were otherwise unremarkable. Plain radiographs and computed tomography scans ± contrast were unremarkable. Cerebrospinal fluid (CSF) from both patients demonstrated eosinophilic pleocytosis, and real-time polymerase chain reaction testing demonstrated Angiostrongylus cantonensis deoxyribonucleic acid in CSF, confirming a diagnosis of neuroangiostrongyliasis. Treatment included glucocorticoid therapy, ± anthelmintic (fenbendazole). Both dogs made a complete recovery. These are the first confirmed cases of autochthonous neuroangiostrongyliasis in canine patients in the United States and the first dogs anywhere to be diagnosed definitively with A cantonensis infection based on real-time polymerase chain reaction testing of CSF. A clinician examining a patient with severe spinal hyperesthesia and a combination of upper and lower motor signs should consider A cantonensis as a differential, especially in endemic areas.

scholarly journals Peran pemeriksaan radiologis pada diagnosis Coronavirus disease 2019

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Budi Yanti ◽  
Ulfa Hayatun
Keyword(s):  
Polymerase Chain Reaction ◽  
Ct Scan ◽  
Real Time ◽  
False Negative ◽  
Ground Glass Opacity ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Lung Abnormalities ◽  
Polymerase Chain ◽  
Radiological Examinations

Abstrak. Coronavirus Disease 19 (COVID-19) telah menjadi pandemi di seluruh dunia dengan angka kejadian yang terus meningkat di beberapa negara. Kecepatan dan ketepatan diagnosis diperlukan untuk mencegah perburukan kondisi pasien. Real-Time Transcription Polymerase Chain Reaction (RT-PCR) sampai saat ini masih menjadi baku emas untuk menegakkan diagnosis COVID-19, namun uji diagnostik ini dilaporkan banyak menunjukkan hasil negatif palsu. Pemeriksaan radiologi berupa foto toraks dan CT-Scan dada banyak dilakukan untuk  menunjang diagnosis COVID-19. Gambaran foto toraks yang paling sering ditemukan adalah konsolidasi, ground-glass opacity (GGO), distribusi bilateral, perifer dan di lobus bawah paru-paru, namun pemeriksaan ini dianggap tidak sensitif untuk menemukan kelainan paru pada tahap awal penyakit. Meskipun demikian, foto toraks dapat digunakan untuk memantau perkembangan kelainan paru akibat COVID-19, salah satunya dengan metode Brixia Score. Pada sisi lain,CT-scan dada dinilai lebih sensitif daripada foto toraks serta mampu menunjukkan kelainan paru tahap awal pada pasien dengan hasil RT-PCR yang negatif. Gambaran pada CT-scan dada umumnya menunjukkan GGO, konsolidasi, crazy-paving stone, dan air bronchogram.  CT-scan dapat mengurangi angka negatif palsu pada RT-PCR dan sebagai alat skrining pada pasien yang dicurigai COVID-19 di lokasi epidemis saat hasil RT-PCR tidak tersedia. Penggunaan pemeriksaan radiologi dan RT-PCR dapat menghemat waktu serta membantu diagnosis dan manajemen COVID-19. Kata Kunci: Pencitraan COVID-19, Radiologi SARS-CoV-2Abstract. Coronavirus Disease 19 (COVID-19) has become a worldwide pandemic with an increasing incidence in several countries. Speed and accuracy of diagnosis are needed to prevent worsening of patient's condition. Real-Time Transcription Polymerase Chain Reaction (RT-PCR) is still a gold standard of COVID-19 diagnosis, however, this test shown false negative results in several case. Radiological examinations, chest X-Ray (CXR) and CT scan, are used to support the diagnosis. The most commonly found in CXR are consolidation, ground-glass opacity (GGO), bilateral distribution, peripheral and in the lower lobe, but this examination is insensitive to find lung abnormalities in early stages of disease. However, CXR can be used to monitor the development of lung abnormalities due to COVID-19, such as the Brixia Score method. On the other hand, CT-Scan is more sensitive than CXR and able to show early lung abnormalities in negative RT-PCR results. CT scan show the presence of GGO, consolidation, crazy-paving stone, and air bronchogram. CT-scanning can reduce the false-negative rate on RT-PCR and become a screening tool in suspected COVID-19 patients at epidemic area where RT-PCR is not available. The use of radiological examinations and RT-PCR can save the time and help in the diagnosis and management of COVID-19.Keywords: COVID-19’s Radiology, Imaging of SARS-CoV-2

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