scholarly journals Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Mayank Gangwar ◽  
Alka Shukla ◽  
Virendra Kumar Patel ◽  
Pradyot Prakash ◽  
Gopal Nath
Keyword(s):  
Isopropyl Alcohol ◽  
Rna Extraction ◽  
Group Testing ◽  
Research Work ◽  
Rt Pcr ◽  
Purification Step ◽  
E Gene ◽  
Qrt Pcr ◽  
Pcr Targeting ◽  
Pooled Samples

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 μL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 μL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49   ng / uL , which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.

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.

Paper or Plastic: RT-PCR of BCR-ABL from Blood Spots Stored and Shipped on Paper

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4566-4566
Author(s):  
Olga Sala Torra ◽  
Lan Beppu ◽  
Susan Branford ◽  
Linda Fletcher ◽  
Gooley Ted ◽  
...  
Keyword(s):  
Room Temperature ◽  
Time Course ◽  
Rna Extraction ◽  
Dried Blood Spots ◽  
Rt Pcr ◽  
Fresh Blood ◽  
Blood Samples ◽  
Blood Spots ◽  
Qrt Pcr ◽  
Sample Age

Abstract In many parts of the world, diagnosis and monitoring of CML patients is limited by the availability and cost of molecular testing. In countries without molecular diagnostic capabilities, blood samples can be shipped to central labs, but this is both hampered by sample degradation, and the high costs of shipping. This study explores the method of directly spotting peripheral blood onto a paper template (dried blood spots), with subsequent shipping, RNA extraction, and BCR-ABL testing. Methods: Blood Spots and Shipment. We received dried blood spots from Australia and African countries by mail or courier, and blood from CML patients from our institution were also used for these experiments. 200μL of blood (PB) was pipetted onto Whatman 503 Protein Saver Cards (PSC; Sigma-Aldrich), where each card contains four 50μL spots. Cards were allowed to dry for at least 24 hours at room temperature. For mailing, PSCs were sealed into glassine envelopes with a packet of desiccant, and then placed inside a mailing envelope following DOT and IATA regulation for shipping non-regulated, exempt human specimens. RNA Extraction from Cards and %BCR-ABL determination. Blood spots were incubated with proteinase K followed by RNA isolation using RNeasy Mini Kits (Qiagen). Extracted RNA was quantified using a NanoDrop spectrometer (Thermo Scientific). %BCR-ABL was determined using the automated Cepheid GeneXpert platform or manual two-step quantitative RT-PCR on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Results: Bench top time course: To test for effects of long transit times on RNA quality, we performed a time course study of cards at room temperature (RT) with 5 samples. For each sample, multiple cards were spotted with PB. The cards were then allowed to sit at RT for predetermined amounts of time, up to 42 days, before extracting RNA. We measured RNA integrity for one of the specimens (CML # 5) and found rapid degradation with the RIN number going from 8.7 for the fresh blood to 2.8 after 28 days on the card. However the amplification for both BCR-ABL and ABL differed less than one cycle between the fresh blood and the last time point by manual qRT-PCR (BCR-ABL Ct = 23.63 for fresh blood and 24.06 for day 28 PSC; ABL Ct = 26.69 for fresh blood and 27.64 for day 28 PSC). Figure 1 shows the results of the time course experiment for the 5 samples as a plot of ΔCt versus time in days. BCR-ABL qRT-PCR concordance studies: We compared the %BCR-ABL results obtained in fresh specimen at the institution sending the sample with the %BCR-ABL results we obtained from RNA extracted from PSC using the Cepheid GeneXpert. Paired evaluable results were available for 9 samples with a median WBC = 9.8 x 109/L (range: 3.37x109/L – 85.5x109/L). Samples were 8 to 49 days old at the time of extraction. The amount of RNA input into the GeneXpert reaction ranged from 38.75ng to 1μg. The %BCR-ABL detected ranged from 0.37% to 27% (see Table). The mean absolute difference between fresh blood and PSC BCR-ABL% is 2%; the relative mean percent change for BCR-ABL, using fresh blood as the reference is 13.1% (S.D., 31.2), P = 0.24. Conclusions and future directions: Dried blood spots are relatively inexpensive method to transport blood that preserves enough RNA stability to allow highly accurate BCR-ABL detection, when compared to results performed on an identical platform using fresh peripheral blood samples. Further studies are undergoing to accurately determine the sensitivity of this method and the feasibility of using regular mail for inexpensive transport of specimens. Table 1IDWBC (1000/μL)Sample Age at Spotting (Days)Sample Age at RNA extraction (Days)RNA ng/μlVolume GeneXPert (μL)Paper %BCR-ABL (IS)GeneXpertFresh Blood % BCR-ABL (IS) GeneXpertI1na010426349naI224.101311092745I38009181544naI47.4285102.4*3.1I55.50495241.92I63.61307.4225912I785.5130102102439I812.212912.415128.8I9na1281.5250.37*0.71I103.370273257.85.7I1115.912731102325I126.612714.415na2.3 *%BCR-ABL was manually calculated due to late ABL Cts because of low starting material. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Self-collected gargle specimen as a patient-friendly sample collection method for COVID-19 diagnosis in a population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

Quantitative Real-Time RT-PCR (qRT-PCR) of Zebrafish Transcripts: Optimization of RNA Extraction, Quality Control Considerations, and Data Analysis

2009 ◽  
Vol 2009 (10) ◽  
pp. pdb.prot5314-pdb.prot5314 ◽  
Author(s):  
C.-C. Lan ◽  
R. Tang ◽  
I. Un San Leong ◽  
D. R. Love
Keyword(s):  
Quality Control ◽  
Data Analysis ◽  
Real Time ◽  
Rna Extraction ◽  
Rt Pcr ◽  
Qrt Pcr

scholarly journals Pooling of Nasopharyngeal Swab Specimens for SARS-CoV-2 detection by RT-PCR

2020 ◽  
Author(s):  
Ignacio Torres ◽  
Eliseo Albert ◽  
David Navarro
Keyword(s):  
Large Population ◽  
Rdrp Gene ◽  
Contact Tracing ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Case Identification ◽  
E Gene ◽  
Sample Pooling ◽  
Community Transmission ◽  
Pcr Targeting

ABSTRACTSystematic testing of large population groups by RT-PCR is mandatory to Covid-19 case identification and contact tracing in order to minimize the likelyhood of resurgence in contagion. Sample pooling for RT-PCR has been effectively used to detect community transmission of SARS CoV-2. Nevertheless, this procedure may decrease the sensitivity of RT-PCR assays due to specimen dilution. We evaluated the efficacy of this strategy for diagnosis of Covid-19 using a sensitive commercially-available RT-PCR targeting SARS CoV-2 E and RdRp genes in a single reaction. A total of 20 mini-pools containing either 5 (n=10) or 10 (n=10) nasopharyngeal exudates collected in universal transport medium were made, each of which including a unique positive NP specimen. Positive specimens yielding CT <32 for the E gene (6 out of 10) or <35.2 for the RdRP gene (7 out of 10) were detected in mini-pools of both sizes. In contrast, most NP samples displaying CTs > 35.8 for the E gene or 35.7 for the RdRP gene remained undetected in mini-pools of 5 specimens (3/4 and 2/3, respectively) or in mini-pools of 10 samples (4/4 and 3/3, respectively.

scholarly journals Self-collected gargle specimen as a patient-friendly sample collection method for COVID-19 diagnosis in a population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

Self-collected gargle as a patient-friendly sample collection method for COVID-19 diagnosis in population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with limitation of sample collection method that requires trained healthcare workers to perform and cause discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days on room temperature storage. The developed method was validated for use on multiple RNA extraction kit and commercially available COVID-19 RT-PCR kits. Our developed method achieved sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35) with 95.16% of patients prefer the self-collected gargle method.

scholarly journals Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

2010 ◽  
Vol 76 (13) ◽  
pp. 4318-4326 ◽  
Author(s):  
Sandhya Parshionikar ◽  
Ian Laseke ◽  
G. Shay Fout
Keyword(s):  
Reverse Transcriptase ◽  
Rna Extraction ◽  
Enteric Viruses ◽  
Health Concern ◽  
Norwalk Virus ◽  
Propidium Monoazide ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Qrt Pcr ◽  
Treated Drinking Water

ABSTRACT Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

scholarly journals Lessons from applied large-scale pooling of 133,816 SARS-CoV-2 RT-PCR tests

2021 ◽  
Vol 13 (589) ◽  
pp. eabf2823 ◽  
Author(s):  
Netta Barak ◽  
Roni Ben-Ami ◽  
Tal Sido ◽  
Amir Perri ◽  
Aviad Shtoyer ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Group Testing ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Open Questions ◽  
Assay Performance ◽  
Theoretical Predictions ◽  
Polymerase Chain ◽  
The Impact

Pooling multiple swab samples before RNA extraction and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis has been proposed as a strategy to reduce costs and increase throughput of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests. However, reports on practical large-scale group testing for SARS-CoV-2 have been scant. Key open questions concern reduced sensitivity due to sample dilution, the rate of false positives, the actual efficiency (number of tests saved by pooling), and the impact of infection rate in the population on assay performance. Here, we report an analysis of 133,816 samples collected between April and September 2020 and tested by Dorfman pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the frequently changing prevalence (0.5 to 6%). We observed pooling efficiency and sensitivity that exceeded theoretical predictions, which resulted from the nonrandom distribution of positive samples in pools. Overall, our findings support the use of pooling for efficient large-scale SARS-CoV-2 testing.

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