scholarly journals A new qualitative RT-PCR assay detecting SARS-CoV-2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marco Favaro ◽  
Walter Mattina ◽  
Enrico Salvatore Pistoia ◽  
Roberta Gaziano ◽  
Paolo Di Francesco ◽  
...  
Keyword(s):  
Internal Control ◽  
Complete Agreement ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Extraction Step ◽  
Two Samples ◽  
Critical Issues ◽  
Microbiological Laboratory ◽  
The One

AbstractThe world is facing an exceptional pandemic caused by SARS-CoV-2. To allow the diagnosis of COVID-19 infections, several assays based on the real-time PCR technique have been proposed. The requests for diagnosis are such that it was immediately clear that the choice of the most suitable method for each microbiology laboratory had to be based, on the one hand, on the availability of materials, and on the other hand, on the personnel and training priorities for this activity. Unfortunately, due to high demand, the shortage of commercial diagnostic kits has also become a major problem. To overcome these critical issues, we have developed a new qualitative RT-PCR probe. Our system detects three genes—RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N)—and uses the β-actin gene as an endogenous internal control. The results from our assay are in complete agreement with the results obtained using a commercially available kit, except for two samples that did not pass the endogenous internal control. The coincidence rate was 0.96. The LoD of our assay was 140 cp/reaction for N and 14 cp/reaction for RdRp and E. Our kit was designed to be open, either for the nucleic acid extraction step or for the RT-PCR assay, and to be carried out on several instruments. Therefore, it is free from the industrial production logics of closed systems, and conversely, it is hypothetically available for distribution in large quantities to any microbiological laboratory. The kit is currently distributed worldwide (called MOLgen-COVID-19; Adaltis). A new version of the kit for detecting the S gene is also available.

scholarly journals A New Qualitative RT-PCR Assay Detecting SARS-CoV-2

2021 ◽  
Author(s):  
Marco Favaro ◽  
Walter Mattina ◽  
Enrico Salvatore Pistoia ◽  
Roberta Gaziano ◽  
Paolo Di Francesco ◽  
...  
Keyword(s):  
Internal Control ◽  
Complete Agreement ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Extraction Step ◽  
Two Samples ◽  
Critical Issues ◽  
Microbiological Laboratory ◽  
Closed Systems

Abstract Purpuse: The world is facing an exceptional pandemic caused by SARS-CoV-2. To allow the diagnosis of COVID infection, several assays based on the real-time PCR technique have been proposed. The requests for diagnosis are such that it was immediately clear that the choice of the most suitable method for each microbiology laboratory had to be based on one hand, the availability of materials, on the other hand, the personnel and training priorities for this activity. Unfortunately, due to high demand, the shortage of commercial diagnostic kits has also become a major problem. To overcome these critical issues, we have developed a new qualitative RT-PCR probe based. Methods: Our system detects three genes: RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N), while the b-actin gene was used as an endogenous internal control. Results: The results of our assay show complete agreement with those obtained using a commercially available kit, except for two samples that did not pass the endogenous internal control. The coincidence rate was 0.96. The LoD of our assay was 140 cp/reaction for N and 14 cp/reaction for RdRp and E. Conclusion: Our kit was designed to be open either for the nucleic acid extraction step or on the RT-PCR assay to be carried out on several instruments. Therefore, it is free from the industrial production logics of closed systems and, conversely, it is hypothetically available for distribution in large quantities in any microbiological laboratory. The kit is currently distributed worldwide (called: MOLgen-COVID-19; Adaltis). A new version of the kit detecting S gene also is coming available.

scholarly journals Application of internal control based on recombinant retroviral particles for increasing the PCR assay reliability

2019 ◽  
Vol 64 (4) ◽  
pp. 420-430
Author(s):  
E. G. Fomina ◽  
E. E. Grigorieva ◽  
E. P. Scheslenok ◽  
P. A. Semizhon ◽  
S. V. Tkachev ◽  
...  
Keyword(s):  
Internal Control ◽  
Reverse Transcription ◽  
False Negative ◽  
Molecular Diagnostic ◽  
Egfp Expression ◽  
Probe Design ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Hybridization Probe

Molecular diagnostic tests based on PCR preceded by reverse transcription (RT-PCR) are now used commonly for the detection of viral pathogens with RNA genomes. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by inhibition or inefficient extraction. In the present study a strategy to produce a new type of internal control for RT-PCR based on recombinant retroviral particles is described. Cell clones stably producing retroviral particles were established by transfecting GP+env-AM12 packaging cells with constructed MoMuLV-derived retroviral vector pLneo/gfp and subsequent cultivation on selective medium with G418. The egfp gene was used as a target for primers and hybridization probe design for real-time RT-PCR assay and as a marker for flow cytometry analysis of eGFP expression by transfected cells. The developed internal control is stable and ribonuclease resistant, economical to produce, noninfectious and safe for routine use. It closely mimics the natural virus and could be successfully used to monitor all the stages of RT-PCR, including nucleic acid extraction, RNA reverse transcription and amplification.

scholarly journals A new system in qualitative RT-PCR detecting SARS-CoV-2 in biological samples: an Italian experience

2020 ◽  
Author(s):  
Marco Favaro ◽  
Walter Mattina ◽  
Enrico Salvatore Pistoia ◽  
Roberta Gaziano ◽  
Paolo Di Francesco ◽  
...  
Keyword(s):  
Internal Control ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Italian Experience ◽  
Large Numbers ◽  
Extraction Step ◽  
Closed Systems ◽  
To Come ◽  
Commercial Kits ◽  
Total Agreement

ABSTRACTIn the last moths the world was faced with the pandemic of a new severe acute respiratory syndrome coronavirus (SARS-CoV) and the majority of the Nations have yet to come out of it. Numerous assays have emerged to meet SARS-CoV-2 diagnostic needs. A clear knowledge of these assays’ parameters is essential to choose the proper test by clinical microbiologists. Unfortunately, the latter cannot be the unique criterion that guides test selection as - given the great demand - shortcomings of commercial kits is also a great issue. Aimed by the intention of overcoming both difficulties we have developed a new qualitative RT-PCR probe based for COVID-19 detection. The system detects three genes of SARS-CoV-2: RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) and β-actin gene used as endogenous internal control. The results of our assay show a total agreement with those obtained using a commercially available kit, with the exception of two specimens which did not pass the endogenous internal control. Moreover, our kit was designed to be open either for nucleic acid extraction step or on the RT-PCR assay to be carried out on several instruments. Thus, it is free from the industrial production logics of closed systems and conversely it is hypothetically available for distribution on large numbers in any microbiological laboratories. Presently, the kit is currently distributed worldwide

scholarly journals An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1370-1374 ◽  
Author(s):  
Mohamad Chikh-Ali ◽  
Stewart M. Gray ◽  
Alexander V. Karasev
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Rna Extraction ◽  
Leaf Tissue ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Potato Leaf ◽  
Extraction Step ◽  
First Time ◽  
Virus Testing

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

Development of a TaqMan® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses

2005 ◽  
Vol 124 (1-2) ◽  
pp. 65-71 ◽  
Author(s):  
Boris Pastorino ◽  
Maël Bessaud ◽  
Marc Grandadam ◽  
Severine Murri ◽  
Hugues J. Tolou ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Extraction Step ◽  
Detection And Quantification

scholarly journals Performance of a PCR Assay for Detection of Pneumocystis carinii from Respiratory Specimens

1998 ◽  
Vol 36 (4) ◽  
pp. 979-982 ◽  
Author(s):  
Angela M. Caliendo ◽  
Peter L. Hewitt ◽  
Jessica M. Allega ◽  
Anne Keen ◽  
Kathryn L. Ruoff ◽  
...  
Keyword(s):  
Internal Control ◽  
Fluorescent Antibody ◽  
Pneumocystis Carinii ◽  
High Sensitivity ◽  
Clinical Microbiology Laboratory ◽  
Hybridization Technique ◽  
Acid Extraction ◽  
Pcr Assay ◽  
Simple Method ◽  
Respiratory Specimens

This study evaluates the performance of a PCR assay for the detection of Pneumocystis carinii from respiratory specimens that has been designed for use in the clinical microbiology laboratory. The test includes a simple method for nucleic acid extraction and amplification, a colorimetric probe hybridization technique for detection of amplicons, and an internal control to evaluate for the presence of inhibitors of amplification. Two hundred thirty-two clinical specimens (120 induced-sputum [IS] and 112 bronchoalveolar lavage [BAL] specimens) from 168 patients were tested by both immunofluorescent (direct fluorescent-antibody [DFA]) staining and PCR. Of the 112 BAL specimens, 17 were positive for P. carinii by DFA staining and PCR. An additional two specimens were DFA negative and PCR positive. For BAL specimens, the sensitivity and specificity of PCR compared to DFA were 100 and 98%, respectively. Eighteen IS specimens were positive for P. carinii by DFA, and 27 were positive by PCR. One of the 18 DFA-positive IS specimens was negative by PCR; this patient had just completed therapy for P. cariniipneumonia. Of the 10 specimens that were PCR positive and DFA negative, 4 were from patients who had a subsequent BAL specimen that was positive by DFA and PCR. For IS specimens, the sensitivity of DFA and PCR was 82 and 95%, respectively. The specificity of PCR for IS specimens was 94%. Due to the high sensitivity of PCR for the detection of P. carinii from IS specimens, a PCR-based diagnostic test may be a useful screening test and may alleviate the need for bronchoscopy in some patients.

scholarly journals Detection of SARS-CoV-2 in saliva: implications for specimen transport and storage

2020 ◽  
Author(s):  
Eloise Williams ◽  
Nicole Isles ◽  
Brian Chong ◽  
Katherine Bond ◽  
Yano Yoga ◽  
...  
Keyword(s):  
N Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Two Samples ◽  
Polymerase Chain ◽  
The Stability ◽  
Gamma Irradiated ◽  
And Storage ◽  
Time Point

Saliva has recently been proposed as a suitable specimen for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Use of saliva as a diagnostic specimen may present opportunities for SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) testing in remote and low-resource settings. Determining the stability of SARS-CoV-2 RNA in saliva over time is an important step in determining optimal storage and transport times. We undertook an in vitro study to assess whether SARS-CoV-2 could be detected in contrived saliva samples. The contrived saliva samples comprised 10 ml pooled saliva spiked with gamma-irradiated SARS-CoV-2 to achieve a concentration of 2.58×104 copies ml SARS-CoV-2, which was subsequently divided into 2 ml aliquots comprising: (i) neat saliva; and a 1 : 1 dilution with (ii) normal saline; (iii) viral transport media, and (iv) liquid Amies medium. Contrived samples were made in quadruplicate, with two samples of each stored at either: (i) room temperature or (ii) 4 °C. SARS-CoV-2 was detected in all SARS-CoV-2 spiked samples at time point 0, day 1, 3 and 7 at both storage temperatures using the N gene RT-PCR assay and time point 0, day 1 and day 7 using the Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, USA) RT-PCR assay. The ability to detect SARS-CoV-2 in saliva over a 1 week period is an important finding that presents further opportunities for saliva testing as a diagnostic specimen for the diagnosis of SARS-CoV-2.

Use of an RT-PCR internal control to evaluate viral removal

1997 ◽  
Vol 35 (11-12) ◽  
pp. 461-465 ◽  
Author(s):  
F. Le Guyader ◽  
D. Menard ◽  
E. Dubois ◽  
L. Haugarreau ◽  
H. Kopecka ◽  
...  
Keyword(s):  
Internal Control ◽  
Peracetic Acid ◽  
Quantitative Estimation ◽  
Acid Extraction ◽  
High Concentration ◽  
Pcr Product ◽  
Extraction Step ◽  
Uv Dose ◽  
Very High

We constructed an internal control based on the poliovirus genome and cloned it into a transcription plasmid to obtain a competitor RNA distinguishable by its size from the wild-type PCR product. A known number of copies was introduced into sewage samples before nucleic acid extraction in order to evaluate the reliability of the extraction step and amplification. The final gel was analysed with a densitometer and the number of viral RNA copies present in the sewage was calculated. In vitro experiments were carried out with this internal control with peracetic acid and UV to estimate decontamination efficiency. A very high concentration of peracetic acid was required to eliminate viruses with a moderate UV dose to obtain a negative PCR. This study demonstrates that competitor RNA is useful both in controlling the different reaction steps (extraction and amplification) and inhibitor detection and in providing a quantitative estimation of contamination.

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