Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene Allplex™ 2019-nCoV assay

Abstract Background The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. Results Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at − 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. Conclusion We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.

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Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene AllplexTM 2019-nCoV assay

10.21203/rs.3.rs-86922/v1 ◽

2020 ◽

Author(s):

Wesley Freppel ◽

Natacha Merindol ◽

Fabien Rallu ◽

Marco Bergevin

Keyword(s):

Detection Rate ◽

Rna Extraction ◽

Free Water ◽

Proteinase K ◽

N Gene ◽

False Negatives ◽

Infected People ◽

Planetary Scale ◽

Testing Method ◽

Viral Genes

Abstract The fight against the COVID-19 pandemic has created an urgent need to detect and isolate infected people. The challenge for clinical laboratories has been finding a high throughput, cheap, and efficient testing method in the context of extraction reagent shortages on a planetary scale. To answer this need, we studied SARS-CoV-2 detection in nasopharyngeal swabs stored in UTM (Universal Transport Media) or RNAse-free water by rRT-PCR with the Seegene Allplex TM 2019-nCoV assay without RNA extraction. Optimal results were obtained with 1/2 dilution for swabs in RNAse free water (30/30 detected) and 1/5 dilution for swabs in UTM (29/30 detected) followed by thermal lysis. In addition, a proteinase K (PK) treatment allows a significant reduction of invalid results and increases sensitivity for detection of low viral load specimens. In a panel of 90 known positives with all 3 viral genes present and N gene Ct values from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a panel of 60 low positives with only the N gene detectable at Ct values > 30, the detection rate was 76.7% with PK vs 53.3% without it and the invalid rate fell off from 18.3% to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, however false negatives become frequent above this range. Finally, we show that swabs should be stored at -70 o C rather than 4 o C when testing cannot be performed within 72 hours of collection when laboratories are overwhelmed.

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Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638718805534 ◽

2018 ◽

Vol 30 (6) ◽

pp. 807-812 ◽

Cited By ~ 2

Author(s):

Ashley C. Weiser ◽

Korakrit Poonsuk ◽

Sarah A. Bade ◽

Phillip C. Gauger ◽

Marisa Rotolo ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Real Time Pcr ◽

False Negative ◽

Rna Extraction ◽

Free Water ◽

Negative Control ◽

Respiratory Syndrome Virus ◽

Sample Handling ◽

Oral Fluids

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate–phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

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False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

10.20944/preprints202009.0450.v2 ◽

2020 ◽

Author(s):

Marcelo Fruehwirth ◽

Açucena Veleh Rivas ◽

Andressa Faria Rahyn Fitz ◽

Aline Cristiane Cechinel Assing Batista ◽

Cleypson Vinicius Silveira ◽

...

Keyword(s):

Internal Control ◽

False Negative ◽

Rna Extraction ◽

N Gene ◽

Gold Standard Method ◽

Negative Results ◽

False Negative Results ◽

Average Variation ◽

Wrong Diagnosis ◽

Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

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Relative Sensitivity of Common Target Genes For The Detection of SARS-Cov-2 In Real Time-PCR

10.21203/rs.3.rs-1118093/v1 ◽

2021 ◽

Author(s):

Reza Valadan ◽

Soheila Golchin ◽

Reza Alizadeh-Navaei ◽

Mohammadreza Haghshenas ◽

Mehryar Zargari ◽

...

Keyword(s):

Real Time ◽

Target Genes ◽

False Negative ◽

Rna Extraction ◽

Host Cells ◽

N Gene ◽

Clinical Samples ◽

Human Beings ◽

Gene Expressions ◽

Mazandaran Province

Abstract SARS-CoV-2(COVID-19) currently is the main cause of the severe acute respiratory disease and fatal outcomes in human beings worldwide. Several genes are used as targets for the detection of SARS-CoV-2, including the RDRP, N, and E genes. The present study aimed to determine the RDRP, N, and E genes expressions of SARS-CoV- 2 in clinical samples. For this purpose, 100 SARS-CoV-2 positive samples were collected from diagnostic laboratories of Mazandaran province, Iran. After RNA extraction, the real time RT-PCR assay was performed for differential gene expressions’ analysis of N, E, and RDRP. The CT values for N, RDRP, and E targets of 100 clinical samples for identifying SARS-CoV-2 were then evaluated using qRT-PCR. This result suggests N gene as a potential target for the detection of the SARS‐CoV‐2, since it was observed to be highly expressed in the nasopharyngeal or oropharynges of COVID-19 patients (P < 0.0001). Herein, we showed that SARS-CoV- 2 genes were differentially expressed in the host cells. Therefore, to reduce obtaining false negative results and to increase the sensitivity of the available diagnostic tests, the target genes should be carefully selected based on the most expressed genes in the cells.

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False-Negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

10.20944/preprints202009.0450.v1 ◽

2020 ◽

Author(s):

Marcelo Fruehwirth ◽

Açucena Veleh Rivas ◽

Andressa Faria Rahyn Fitz ◽

Aline Cristiane Cechinel Assing Batista ◽

Cleypson Vinicius Silveira ◽

...

Keyword(s):

Internal Control ◽

False Negative ◽

Rna Extraction ◽

N Gene ◽

Gold Standard Method ◽

Negative Results ◽

False Negative Results ◽

Average Variation ◽

Wrong Diagnosis ◽

Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®) for extraction. rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Ct between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811Ct, 3.840Ct, and 3.842Ct, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.8% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

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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

10.21203/rs.3.rs-153817/v1 ◽

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.

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Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

10.1101/2020.06.12.147819 ◽

2020 ◽

Cited By ~ 4

Author(s):

Rob J. Dekker ◽

Wim A. Ensink ◽

Selina van Leeuwen ◽

Han Rauwerda ◽

Timo M. Breit

Keyword(s):

False Positive ◽

False Negative ◽

Rna Extraction ◽

N Gene ◽

Rt Pcr ◽

P Gene ◽

Reverse Primer ◽

Probe Set ◽

Pcr Test ◽

Test Control

ABSTRACTTo battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy.We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as “2019-nCoV not detected”, whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as “Invalid Result” and the procedure should be repeated.In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

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A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity

Microorganisms ◽

10.3390/microorganisms8071064 ◽

2020 ◽

Vol 8 (7) ◽

pp. 1064 ◽

Cited By ~ 3

Author(s):

Sara Petrillo ◽

Giovanna Carrà ◽

Paolo Bottino ◽

Elisa Zanotto ◽

Maria Chiara De Santis ◽

...

Keyword(s):

Internal Control ◽

False Negative ◽

Rna Extraction ◽

Simultaneous Detection ◽

High Sensitivity ◽

N Gene ◽

Qrt Pcr ◽

P Gene ◽

Global Pandemic ◽

Pcr Method

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.

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Routine blood tests as a potential diagnostic tool for COVID-19

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0398 ◽

2020 ◽

Vol 58 (7) ◽

pp. 1095-1099 ◽

Cited By ~ 34

Author(s):

Davide Ferrari ◽

Andrea Motta ◽

Marta Strollo ◽

Giuseppe Banfi ◽

Massimo Locatelli

Keyword(s):

Blood Test ◽

Hematological Parameters ◽

False Negative ◽

White Blood Cells ◽

Turnaround Time ◽

Test Analysis ◽

Infected People ◽

Reactive Protein ◽

Routine Blood ◽

Glutamyl Transpeptidase

AbstractObjectivesThe outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to date, the epidemic has gradually spread to 209 countries worldwide with more than 1.5 million infected people and 100,000 deaths. Amplification of viral RNA by rRT-PCR serves as the gold standard for confirmation of infection, yet it needs a long turnaround time (3–4 h to generate results) and shows false-negative rates as large as 15%–20%. In addition, the need of certified laboratories, expensive equipment and trained personnel led many countries to limit the rRT-PCR tests only to individuals with pronounced respiratory syndrome symptoms. Thus, there is a need for alternative, less expensive and more accessible tests.MethodsWe analyzed the plasma levels of white blood cells (WBCs), platelets, C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), alkaline phosphatase and lactate dehydrogenase (LDH) of 207 patients who, after being admitted to the emergency room of the San Raffaele Hospital (Milan, Italy) with COVID-19 symptoms, were rRT-PCR tested. Of them, 105 tested positive, whereas 102 tested negative.ResultsStatistically significant differences were observed for WBC, CRP, AST, ALT and LDH. Empirical thresholds for AST and LDH allowed the identification of 70% of either COVID-19-positive or -negative patients on the basis of routine blood test results.ConclusionsCombining appropriate cutoffs for certain hematological parameters could help in identifying false-positive/negative rRT-PCR tests. Blood test analysis might be used as an alternative to rRT-PCR for identifying COVID-19-positive patients in those countries which suffer from a large shortage of rRT-PCR reagents and/or specialized laboratory.

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Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab039 ◽

2021 ◽

Author(s):

Ron M Kagan ◽

Amy A Rogers ◽

Gwynngelle A Borillo ◽

Nigel J Clarke ◽

Elizabeth M Marlowe

Keyword(s):

Return To Work ◽

Screening Program ◽

False Negative ◽

N Gene ◽

Rt Pcr ◽

Pooled Testing ◽

Specimen Collection ◽

Asymptomatic Patients ◽

Nasal Swabs ◽

The Impact

Abstract Background The use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for SARS-CoV-2 RT-PCR can decrease the use of PPE and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort. Methods Self-collected anterior nasal swabs from employee return to work programs were tested using the Quest Diagnostics SARS-CoV-2 RT-PCR EUA. The Ct values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of HCP-collected specimens from patients with and without COVID-19 symptoms. Results Among 47,923 participants, 1.8% were positive. RP failed to amplify for 13/115,435 (0.011%) specimens. The median (IQR) Cts were 32.7 (25.0-35.7) for N1 and 31.3 (23.8-34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic, but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1,268 (5.2%) specimens but only a single false-negative result. Conclusions Unobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population as the likelihood of false negative results is very low due when using a sensitive, dual-target methodology.

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