scholarly journals Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255690
Author(s):  
Jennifer R. Hamilton ◽  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Enrique Lin-Shiao ◽  
C. Kimberly Tsui ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rna Extraction ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab ◽  
Infection Prevalence ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Rapid Pcr ◽  
Large Populations ◽  
Asymptomatic Individuals

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

scholarly journals Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

2021 ◽  
Author(s):  
Jennifer R. Hamilton ◽  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Enrique Lin-Shiao ◽  
C. Kimberly Tsui ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rna Extraction ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab ◽  
Infection Prevalence ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Rapid Pcr ◽  
Large Populations ◽  
Asymptomatic Individuals

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

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 Evaluation of Three Automated Nucleic Acid Extraction Systems for Identification of Respiratory Viruses in Clinical Specimens by Multiplex Real-Time PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Yoonjung Kim ◽  
Mi-Soon Han ◽  
Juwon Kim ◽  
Aerin Kwon ◽  
Kyung-A Lee
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
False Negative ◽  
Virus Detection ◽  
Turnaround Time ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction ◽  
Negative Results

A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

Research note: Rapid isolation of total RNA and genomic DNA from Hakea actities

2002 ◽  
Vol 29 (8) ◽  
pp. 1015 ◽  
Author(s):  
Michael G. Mason ◽  
Susanne Schmidt
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Genomic Dna ◽  
Rna Extraction ◽  
Nutrient Concentrations ◽  
Cluster Roots ◽  
Acid Extraction ◽  
Native Plant ◽  
Nucleic Acid Extraction ◽  
Total Rna

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

scholarly journals Parallel RNA extraction using magnetic beads and a droplet array

Lab on a Chip ◽  
2015 ◽  
Vol 15 (4) ◽  
pp. 1059-1065 ◽  
Author(s):  
Xu Shi ◽  
Chun-Hong Chen ◽  
Weimin Gao ◽  
Shih-hui Chao ◽  
Deirdre R. Meldrum
Keyword(s):  
Nucleic Acid ◽  
Magnetic Beads ◽  
Rna Extraction ◽  
Lab On A Chip ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Droplet Array

Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device.

scholarly journals Utility of Nucleic Acid Extraction Free COVID-19 Real Time PCR Protocol in Resource Limited Setting: A Pilot Study

2021 ◽  
Author(s):  
Santosh Karade ◽  
Pratik Thosani ◽  
Prashant Patil ◽  
Kavita Bala Anand ◽  
Sourav Sen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pilot Study ◽  
Real Time ◽  
Gold Standard ◽  
Rna Extraction ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Resource Limited

Introduction: Coronavirus Disease (COVID-19), a respiratory infection, caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, Hubei province, China in December 2019. Alarming increase in the number of cases has put tremendous pressure on existing health resources. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a molecular diagnostic method, is considered gold standard for diagnosis of SARS-CoV-2 infection. It involves RNA extraction as the preliminary step. Innovations to cut down cost and time involved in SARS-CoV-2 testing are need of hour. Aim: The aim of this study was to assess the feasibility of Nucleic Acid Extraction Free (NEF) protocol for COVID-19 diagnosis in resource limited settings. Materials and Methods: In this pilot study a panel of 148 Nasopharyngeal (NP) samples was subjected to the novel NEF RT-PCR protocol and results were compared to gold standard RT-PCR on RNA extracted from NP specimen. The cycle threshold value for each target was tabulated in MS Excel Spreadsheet and data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 15.0. Results: Out of 148 collected samples, 120 showed amplification of E and RdRp targets by RNA extraction-based RT-PCR. Overall sensitivity and specificity observed for NEF protocol was 43.94% and 96.42%, respectively. Conclusion: Further refinement in the protocol would be required to improve the sensitivity of NEF protocol and widespread use in laboratories.

scholarly journals Evaluation of commercial methods to separate nucleic acids from intestinal tissues of pigs for diagnosis of porcine epidemic diarrhea

2019 ◽  
Vol 10 (4) ◽  
pp. 477-483
Author(s):  
D. М. Masiuk ◽  
V. S. Nedzvetsky ◽  
A. V. Kokariev ◽  
O. V. Danchuk ◽  
T. O. Vasilenko ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Solid Phase ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Viral Dna ◽  
Tissue Mass ◽  
Porcine Epidemic Diarrhea ◽  
Commercial Kits

The article presents the results of evaluating commercial methods for extracting nucleic acids from pig intestinal tissues for the diagnosis of PED. The study was based on samples of small intestine tissues and faeces from 3–5 day old pigs which died from PED. Nucleic acid extraction was performed using commercial kits with different nucleic acid separation strategies based on: silicon-sorbent; silicate membrane fixed in a microcentrifuge column and magnetic balls. The studies were conducted in two stages. The first was a comparison of the results of the amplification of the obtained nucleic acid extracts from the homogenate of the intestines of piglets by using the above-mentioned commercial kits for the extraction of nucleic acids. For this purpose, samples of homogenate were used which in weight corresponded to the guideline for the application of the test kits. The second step was directed to determining the efficiency of extraction of DNA and RNA from homogenate samples with a weight of 10, 50, 100 and 200 mg. Determination of the optimal methodological strategy of nucleic acid extraction for the diagnosis of porcine epidemic diarrhea by PCR has been investigated. The results of the PCR studies of RNA of the PED virus and a unique pig DNA fragment indicate that the extraction of nucleic acids by commercial kits has different levels of efficiency and depends on different factors. According to the research, it was found that the most important of them are the adsorption capacity of the solid-phase sorbent, its configuration and nature, which binds RNA and DNA molecules, the type of sample from which extraction takes place, its volume, or the tissue mass used for extraction. Based on the obtained results, it has been found that the most effective PED virus RNA extraction is by “ArtBioTech”, “Bio Extract Column”, and “Viral DNA/RNA Extraction Kit”, and pig genomic DNA extraction by the “ArtBioTech” and “Viral DNA / RNA extraction Kit”.

Automated Extraction of Plant Viral RNA Using the Nucleic Acid Instrument Based on Magnetic Nanobeads

2013 ◽  
Vol 303-306 ◽  
pp. 646-649
Author(s):  
Yong Jie Dou ◽  
Xiao Li Zhao ◽  
Jian Sheng Xu ◽  
Cong Liang Deng ◽  
Leng Nie ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Plant Virus ◽  
Rna Extraction ◽  
Viral Rna ◽  
Acid Extraction ◽  
Automated Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Initial Concentration ◽  
Magnetic Nanobeads

In this paper, an automated nucleic acid extraction instrument based on magnetic nanobeads is described. Also, a prototype is designed and constructed. Some plant viral RNA extraction experiments were done using the platform. And the experimental results demonstrated that the instrument could be successfully used for extraction of plant viral RNA，and the extracted RNA was successfully used in an automated PCR assay for the detection of plant virus. The Ct values of CGMMV RNA, LSV RNA and ArMV RNA at the initial concentration were 12.51, 17.81 and 23.48.

scholarly journals Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

2011 ◽  
Vol 77 (13) ◽  
pp. 4336-4343 ◽  
Author(s):  
Akihiko Hata ◽  
Hiroyuki Katayama ◽  
Masaaki Kitajima ◽  
Chettiyappan Visvanathan ◽  
Chea Nol ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Water Samples ◽  
Rna Extraction ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Acid Extraction ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Viral Nucleic Acid

ABSTRACTInhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

scholarly journals Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections

2017 ◽  
Vol 55 (7) ◽  
pp. 2035-2044 ◽  
Author(s):  
Clayton O. Onyango ◽  
Vladimir Loparev ◽  
Shirley Lidechi ◽  
Vinod Bhullar ◽  
D. Scott Schmid ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Nucleic Acid ◽  
Sub Saharan Africa ◽  
Detection Sensitivity ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Content Type ◽  
Herpes Simplex Viruses ◽  
Taqman Array

ABSTRACT Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites ( Balamuthia mandrillaris and Acanthamoeba ), six bacterial pathogens ( Streptococcus pneumonia e, Haemophilus influenzae , Neisseria meningitidis , Mycoplasma pneumoniae , Mycobacterium tuberculosis , and Bartonella ), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease.

Sign in / Sign up

Export Citation Format

Share Document