A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses

2017 ◽  
Vol 247 ◽  
pp. 61-67 ◽  
Author(s):  
Fan Yang ◽  
Guoping Wang ◽  
Wenxing Xu ◽  
Ni Hong
Keyword(s):  
Rna Extraction ◽  
Fruit Trees ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Spin Column

scholarly journals Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
1997 ◽  
Vol 81 (2) ◽  
pp. 222-226 ◽  
Author(s):  
Donald J. MacKenzie ◽  
Morven A. McLean ◽  
Srima Mukerji ◽  
Margaret Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Woody Plants ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Apple Stem ◽  
Spin Column ◽  
Polymerase Chain

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

scholarly journals Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples

2021 ◽  
pp. 114302
Author(s):  
Stephany D. Villota ◽  
Victoria E. Nipaz ◽  
Andrés Carrazco-Montalvo ◽  
Sarah Hernandez ◽  
Jesse J. Waggoner ◽  
...  
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Pcr Detection ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
The Real

scholarly journals Effect of Heat Inactivation for the Detection of Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2) with Reverse Transcription Real Time Polymerase Chain Reaction (rRT-PCR): Evidence from Ethiopian Study

2021 ◽  
Author(s):  
Belete Woldesemayat Hailemariam ◽  
Gebremedihin Gebremicael ◽  
Kidist Zealias ◽  
Amelework Yilma ◽  
Sisay Adane ◽  
...  
Keyword(s):  
Pearson Correlation ◽  
False Negative ◽  
Rna Extraction ◽  
Pcr Detection ◽  
N Gene ◽  
Heat Inactivation ◽  
P Value ◽  
Rt Pcr ◽  
Specimen Handling ◽  
Ct Value

Abstract Background: Coronavirus disease 2019 (COVID-19) specimen handling needs a major concern due to the virus has a potential of easily transmittable to health care workers and laboratory personnel. Heat inactivation before nucleic acid isolation might permit safe testing, even though, the effect of heat inactivation on SARS-CoV-2 RT-PCR detection results needs to be determined. Methods: An experimental study was conducted in Ethiopian Public Health Institute (EPHI) from September 25 to October 15, 2020. A total of 188 Oro-pharyngeal swabs were collected from COVID-19 suspected cases, referred to EPHI for SARS COV-2 testing during the study period. One group of the sample was inactivated at 56 °C heat for 30 min, and the other group was stored at 4°C for a similar period of time. RNA extraction and detection were done by DAAN Gene extraction and detection kit. Abbott m2000 RT-PCR was used for amplification and detection. Data analysis was done by using SPSS version 23.0; Chi-square and Pearson correlation test for qualitative and semi-quantitative analysis were used. P-value < 0.05 was considered as statistically significant.Results: Out of 188 total samples, 117 (62.2 %) and 118 (62.8%) were positive for ORF1a/b and N gene respectively before inactivation. Whereas after inactivation, 111 (59 %) was ORF1a/b and 116 (61.7 %) was N gene positive. Rate of positivity between groups was not statistically significant (p>0.05). The mean CT value difference between the two groups of ORF1a/b gene and N gene was 0.042 (95 % CI, -0.247- 0.331; t= 0.28; p = 0.774) and 0.38 (95% CI, 0.097 - 0.682; t =2.638; p = 0.010) respectively.Conclusion: Heat inactivation at 56 ℃ for 30 min has not statistically significant effect for the qualitative rRT-PCR detection of SARS-CoV-2. However, the finding also showed that there was statistically significant CT value increment after heat inactivation compared to untreated samples. Therefore, false-negative results with high CT value results (CT > 35) were found to be the challenge of this protocol. Hence alternative inactivation methods should be investigated and further studies should be considered.

scholarly journals Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection

2020 ◽  
Vol 132 ◽  
pp. 104587
Author(s):  
Ruby Barza ◽  
Parul Patel ◽  
Linda Sabatini ◽  
Kamaljit Singh
Keyword(s):  
Rna Extraction ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Sample Processing ◽  
Processing Step

Comparison of RNA extraction methods for RT-PCR detection of Coconut cadang-cadang viroid variant in orange spotting oil palm leaves

2016 ◽  
Vol 38 (3) ◽  
pp. 382-388 ◽  
Author(s):  
Nur D. Roslan ◽  
Ong A. Meilina ◽  
Intan-Nur A. Mohamed-Azni ◽  
Idris A. Seman ◽  
Shamala Sundram
Keyword(s):  
Oil Palm ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Rna Extraction Methods

scholarly journals A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

Healthcare ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 37
Author(s):  
Sherif A. El-Kafrawy ◽  
Mai M. El-Daly ◽  
Ahmed M. Hassan ◽  
Reham M. Kaki ◽  
Adel M. Abuzenadah ◽  
...  
Keyword(s):  
Rna Virus ◽  
Direct Method ◽  
Rna Extraction ◽  
Pcr Detection ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Direct Pcr ◽  
Rdrp Region ◽  
Positive Strand Rna

Introduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies. Methods: we optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes. Results: our direct detection protocol showed 100% concordance with the standard protocol with an average Ct value difference of 4.38 for the E region and 3.85 for the RdRp region. Conclusion: the direct PCR technique was found to be a reliable and sensitive method that can be used to reduce the time and cost of the assay by removing the need for RNA extraction. It enables the use of the assay in research, diagnostics, and screening for COVID-19 in regions with fewer economic resources, where supplies are more limited allowing for wider use for screening.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

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