scholarly journals Evaluation of Three Automated Extraction Systems for the Detection of SARS-CoV-2 from Clinical Respiratory Specimens

Life ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 68
Author(s):  
Ho-Jae Lim ◽  
Hye-Soo Jung ◽  
Min-Young Park ◽  
Young-Hyun Baek ◽  
Balaji Kannappan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Large Scale ◽  
Quantitative Polymerase Chain Reaction ◽  
Acid Extraction ◽  
Viral Pathogen ◽  
Infectious Dose ◽  
Nucleic Acid Isolation ◽  
Thermo Fisher Scientific ◽  
Clinical Detection ◽  
Respiratory Specimens

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly contagious and causes coronavirus disease 2019 (COVID-19). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the most accurate and reliable molecular assay to detect active SARS-CoV-2 infection. However, a rapid increase in test subjects has created a global bottleneck in testing capacity. Given that efficient nucleic acid extraction greatly affects reliable and accurate testing results, we compared three extraction platforms: MagNA Pure 96 DNA and Viral NA Small Volume kit on MagNA Pure 96 (Roche, Basel, Switzerland), careGENETM Viral/Pathogen HiFi Nucleic Acid Isolation kit (WELLS BIO Inc., Seoul, Korea) on KingFisher Flex (Thermo Fisher Scientific, Rocklin, CA, USA), and SGRespiTM Pure kit (Seegene Inc., Seoul, Korea) on Maelstrom 9600 (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). RNA was extracted from 245 residual respiratory specimens from the different types of samples (i.e., NPS, sputum, and saliva) using three different kits. The 95% limits of detection of median tissue culture infectious dose per milliliter (TCID50/mL) for the MagNA Pure 96, KingFisher Flex, and Maelstrom 9600 were 0.37–3.15 × 101, 0.41–3.62 × 101, and 0.33–1.98 × 101, respectively. The KingFisher Flex platform exhibited 99.2% sensitivity and 100% specificity, whereas Maelstrom 9600 exhibited 98.3–100% sensitivity and 100% specificity. Bland–Altman analysis revealed a 95.2% concordance between MagNA Pure 96 and KingFisher Flex and 95.4% concordance between MagNA Pure 96 and Maelstrom 9600, indicating that all three platforms provided statistically reliable results. This suggests that two modifying platforms, KingFisher Flex and Maelstrom 9600, are accurate and scalable extraction platforms for large-scale SARS-CoV-2 clinical detection and could help the management of COVID-19 patients.

scholarly journals SARS-CoV-2 sample-to-answer nucleic acid testing in a tertiary care emergency department: evaluation and utility

2020 ◽  
Author(s):  
Pia Jokela ◽  
Anu E Jääskeläinen ◽  
Hanna Jarva ◽  
Tanja Holma ◽  
Maarit J Ahava ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Emergency Departments ◽  
Large Scale ◽  
Tertiary Care ◽  
Reference Method ◽  
Preliminary Evaluation ◽  
Specificity And Sensitivity ◽  
Department Evaluation ◽  
Test Result ◽  
Respiratory Specimens

AbstractRapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert® Xpress SARS-CoV-2 and Mobidiag Novodiag® Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag® test in tertiary care emergency departments was assessed. In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert®, whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag®. Rapid SARS-CoV-2 testing with Novodiag® was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag® and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag® and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag®, but positive with the reference method with late Ct values. On average, a test result using Novodiag® was available nearly 8 hours earlier than that obtained with the large-scale PCR tests. While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.

scholarly journals SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

2020 ◽  
Author(s):  
Chantal B.F. Vogels ◽  
Anne E. Watkins ◽  
Christina A. Harden ◽  
Doug Brackney ◽  
Jared Shafer ◽  
...  
Keyword(s):  
Supply Chain ◽  
Nucleic Acid ◽  
Large Scale ◽  
Limit Of Detection ◽  
Proteinase K ◽  
Acid Extraction ◽  
Sample Collection ◽  
Reverse Transcription Pcr ◽  
August 15Th ◽  
Frequent Sampling

Current bottlenecks for improving accessibility and scalability of SARS-CoV-2 testing include diagnostic assay costs, complexity, and supply chain shortages. To resolve these issues, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration on August 15th, 2020. The critical component of our approach is to use saliva instead of respiratory swabs, which enables non-invasive frequent sampling and reduces the need for trained healthcare professionals during collection. Furthermore, we simplified our diagnostic test by (1) not requiring nucleic acid preservatives at sample collection, (2) replacing nucleic acid extraction with a simple proteinase K and heat treatment step, and (3) testing specimens with a dualplex quantitative reverse transcription PCR (RT-qPCR) assay. We validated SalivaDirect with reagents and instruments from multiple vendors to minimize the risk for supply chain issues. Regardless of our tested combination of reagents and instruments from different vendors, we found that SalivaDirect is highly sensitive with a limit of detection of 6-12 SARS-CoV-2 copies/μL. When comparing SalivaDirect to paired nasopharyngeal swabs using the authorized ThermoFisher Scientific TaqPath COVID-19 combo kit, we found high agreement in testing outcomes (>94%). In partnership with the National Basketball Association (NBA) and Players Association, we conducted a large-scale (n = 3,779) SalivaDirect usability study and comparison to standard nasal/oral tests for asymptomatic and presymptomatic SARS-CoV-2 detection. From this cohort of healthy NBA players, staff, and contractors, we found that 99.7% of samples were valid using our saliva collection techniques and a 89.5% positive and >99.9% negative test agreement to swabs, demonstrating that saliva is a valid and noninvasive alternative to swabs for large-scale SARS-CoV-2 testing. SalivaDirect is a flexible and inexpensive ($1.21-$4.39/sample in reagent costs) option to help improve SARS-CoV-2 testing capacity. Register to become a designated laboratory to use SalivaDirect under our FDA EUA on our website: publichealth.yale.edu/salivadirect/.

scholarly journals Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens

2012 ◽  
Vol 84 (6) ◽  
pp. 906-911 ◽  
Author(s):  
C. Mengelle ◽  
J.-M. Mansuy ◽  
K. Sandres-Sauné ◽  
C. Barthe ◽  
J. Boineau ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Extraction System ◽  
Acid Extraction ◽  
Prospective Evaluation ◽  
Nucleic Acid Extraction ◽  
Respiratory Specimens

scholarly journals Impact of two different commercial DNA extraction methods on BK virus viral load

2016 ◽  
Vol 31 (1) ◽  
Author(s):  
Massimiliano Bergallo ◽  
Ilaria Galliano ◽  
Elisa Loiacono ◽  
Francesca Ferro ◽  
Paola Montanari ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
Dna Extraction ◽  
Quantitative Polymerase Chain Reaction ◽  
Extraction Procedure ◽  
Bk Virus ◽  
Human Polyomavirus ◽  
Acid Extraction ◽  
Seroprevalence Rate ◽  
The Impact

Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. <br />Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France) and manual QIAGEN extraction (QIAGEN Hilden, Germany). BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. <br />Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit), for the extraction of BK virus from serum and urine specimens.

scholarly journals Co-occurrence of SARS-CoV-2 and Respiratory Pathogens in the Frail Elderly

2020 ◽  
Author(s):  
Alan Wolfe ◽  
David Baunoch ◽  
Dakun Wang ◽  
Ryan Gnewuch ◽  
Xinhua Zhao ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Nucleic Acid ◽  
Assisted Living ◽  
Klebsiella Pneumonia ◽  
Assisted Living Facilities ◽  
Respiratory Pathogens ◽  
Total Nucleic Acid ◽  
Detection Rates ◽  
Viral Pathogen ◽  
Nucleic Acid Isolation

AbstractBackgroundElderly SARS-CoV-2 patients are associated with higher hospitalization and mortality. Co-infection is critical in the severity of respiratory diseases. It is largely understudied for SARS-CoV-2.MethodsBetween March 24th and April 27th, 2020, nasopharyngeal and oropharyngeal swabs from 3,348 patients from nursing homes and assisted living facilities in 22 states in the US were tested by Capstone Healthcare for SARS-CoV-2, 24 other respiratory viruses, and 8 respiratory bacteria. Total nucleic acid was extracted with MagMAX™ Viral/Pathogen Ultra nucleic acid isolation kit. SARS-Co-V-2 was detected with the CDC 2019-novel coronavirus (2019-nCoV) diagnostic panel. Total nucleic acid was pre-amplified before analysis for other respiratory pathogens with Taqman OpenArray™ Respiratory Tract Microbiota Plate.ResultsPatients’ mean age was 76.9 years. SARS-CoV-2 was detected in 1,413 patients (42.2%). Among them, 1,082 (76.6%) and 737 (43.7%) patients were detected with at least one bacterium or another virus, respectively. SARS-CoV-2-positive patients were more likely to have bacterial co-occurrences (76.6%) than SARS-CoV-2-negative patients (70.0%) (p<0.0001). The most common co-occurring bacteria were Staphylococcus aureus and Klebsiella pneumonia, detected in 55.8% and 40.1% SARS-CoV-2-positive patients, respectively. Staphylococcus aureus was associated with SARS-CoV-2, with higher detection rates in SARS-CoV-2-positive patients (55.8%) than SARS-CoV-2-negative patients (46.2%) (p<0.0001). Human herpes virus 6 (HHV6) also was common and associated with SARS-CoV-2, with higher detection rates in SARS-CoV-2-positive patients (26.6%) than SARS-CoV-2-negative patients (19.1%) (p<0.0001).ConclusionsSARS-CoV-2-positive patients are more likely to be positive for certain respiratory bacteria and viruses. This observation may help explain high hospitalization and mortality rates in older patients.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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.

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