Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus

Jiang, Yushen; Zhang, Shanming; Qin, Hong; Meng, Shuai; Deng, Xian-Yu; He, Lin; Xin, Xiaoliang; Liang, Yuxin; Chen, Bowen; Cui, Yan; Su, YiHeng; Liang, Pei; GuangZhi, Zhou; Hu, Hongbo

doi:10.1186/s40001-021-00608-5

Cited by 13 publications

(10 citation statements)

References 17 publications

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“…Previous experiments done in our laboratory using the NEB’s WarmStart Colorimetric LAMP 2X Master Mix with primers targeting genes N and E of SARS-CoV-2 could not achieve a limit of detection below 50 copies per μL (data not shown). The limit of detection of RT-PCR tests using purified samples varies between 0.08 19 and 14 20 copies per μL 21 – 23 . Comparing our LoD with other molecular tests using a direct swab, our limit of detection was 25,000 copies per mL when the LoD of the commercial tests were 60,000–540,000 NDU per mL 24 —viral RNA copies/mL is equivalent to genome copy equivalents/mL (GCE/mL) or nucleic acid detectable units/mL (NDU/mL) 25 .…”

Section: Discussionmentioning

confidence: 99%

Development of an optimized colorimetric RT-LAMP for SARS-CoV-2 assay with enhanced procedure controls for remote diagnostics

Raddatz

Kim

Imamura

et al. 2022

Sci Rep

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The coronavirus pandemic accentuated the need for molecular diagnostic tests. A technique highly used to this end is the Polymerase Chain Reaction (PCR)—a sensitive and specific technique commonly used as the gold standard for molecular diagnostics. However, it demands highly trained personnel and high-maintenance equipment and is relatively time-consuming. An alternative is the Loop-Mediated Isothermal Amplification (LAMP) technique, which doesn’t need sample purification or expensive equipment, and is similar to PCR when compared in sensitivity and specificity. In this paper, we developed an optimized colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) Point-of-Care test using a portable device to diagnose COVID-19. Variables such as concentration of primers, magnesium sulfate, betaine, hydrochloride guanidine, Bst, and temperature of the reactions were tested. We also created a pipetting quality control system—using a combination of dyes—to avoid false negatives due to a lack of samples added to the reaction test tube. Mineral oil was incorporated in the composition of the RT-LAMP reactions to avoid evaporation when a heating lid isn't available. The final RT-LAMP test is tenfold more sensitive when compared to the WarmStart Colorimetric Master mix from New England Biolabs with a sensitivity of 5 copies per μL.

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Section: Discussionmentioning

confidence: 99%

Development of an optimized colorimetric RT-LAMP for SARS-CoV-2 assay with enhanced procedure controls for remote diagnostics

Raddatz

Kim

Imamura

et al. 2022

Sci Rep

View full text Add to dashboard Cite

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“…A real-time Polymerase Chain Reaction (rtPCR) test was performed in the case of infection either locally or in public clinics using the FDA-approved TaqPath Combo Kit targeting the N2, ORF1Ab, and S genes [12]. Infection was then con rmed by a quantitative test of Anti-N-IgG to the SARS-CoV-2 nucleoprotein using Elecsys® immunoassay (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's instructions.…”

Section: Serological Testingmentioning

confidence: 99%

Serological responses to the BNT-162b2 Pfizer/BioNTech vaccination in twin pregnancies- Comparison between immunization before conception and during pregnancy.

Svirsky

Rabbi

Hamad³

et al. 2023

Preprint

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Objectives Compare the levels of neutralizing Anti-Spike Protein IgG (Anti-S-IgG) during twin pregnancies in women who were immunized by the BNT-62b2-Pfizer/BioNTech vaccine for COVID-19 before conception vs. during pregnancy. Methods In this prospective study, three blood samples were collected from 83 pregnant and 200 age-matched non-pregnant women and subjected to Anti-S-IgG immunodiagnostics. Patient’s medical records were collected from the hospital’s electronic database, including vaccination and PCR test results. Results Of the 83 women with twin pregnancies, 74 were dichorionic diamniotic (DCDA) and 9 were monochorionic diamniotic (MCDA), 49 were vaccinated before conception, 21 were vaccinated during pregnancy, and 13 were non-vaccinated. Nine non-vaccinated women experienced an uneventful pregnancy, one contracted COVID-19 before conception, three became positive during pregnancy, and all three were severely ill and required hospitalization in the 3rd trimester. By contrast, in two (9.5%) of the 21 women who were vaccinated during pregnancy and 7 (14.3%) of the 46 women vaccinated before conception COVID-19 infection during pregnancy caused only mild symptoms. The levels of neutralizing Anti-S-IgG in the 3rd trimester in the women vaccinated before conception were 1,475 Au/mL [IQR 392-3,020] vs. 112 [54–357] (ten-fold lower) in women vaccinated during pregnancy (p < 0.001)). No difference was found between DCDA and MCDA. Third-trimester Anti-S-IgG levels in twin pregnancies were higher compared to non-pregnant women and to gravid women with singletons (according to reported levels). In women vaccinated at 13–16 weeks gestation, neutralizing Anti-S-IgG at 20–22 weeks went up to 372 Au/mL[120–1598], but rapidly dropped to 112[54–357) at 28–30 weeks, (p < 0.001), a faster decline than in women vaccinated before conception, or women with singleton pregnancies, or non-pregnant women (p < 0.001). Infection before conception was associated with having low Anti-S-IgG levels during pregnancy, whereas COVID-19 during pregnancy led to a very high response in the 3rd trimester, as previously reported for singleton pregnancies. Conclusion Lower neutralizing Anti-S-IgG levels were observed in women who were vaccinated during pregnancy compared to those who were vaccinated before conception, and they also declined faster. Vaccinated women who came down with COVID-19 had milder symptoms than unvaccinated women. Thus, a full course of vaccination before conception is recommended. ClinicalTrials.gov Protocol Registration and Results System (PRS) Receipt Release Date: October 4, 2021 ClinicalTrials.gov ID: NCT04595214

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“…Three genes, including E and N genes of SARS-CoV-2, and the human RNAse gene, were detected using the Quant Studio Real-Time PCR system (ThermoFisher Scientific, Waltham, MA, USA) and the SOLIScript 1-step multiplex probe kit (ROX) (SOLIS BIODYNE, USA), according to the manufacturer's protocol. Identification of the E-genewas done for screening, and detection of the N-gene confirmed SARS-CoV-2 infection, while human RNAse was used as control [23]. A cycle threshold value less than 37 was documented as a positive result, and 38 and more was documented as a negative result.…”

Section: Study Populationmentioning

confidence: 99%

Influence of SARS-COV-2 Infection on Cytokine Production by Mitogen-Stimulated Peripheral Blood Mononuclear Cells and Neutrophils in COVID-19 Intensive Care Unit Patients

et al. 2022

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We sought to investigate the influence of SARS-CoV-2 infection on the cytokine profiles of peripheral blood mononuclear cells (PBMCs) and neutrophils from coronavirus disease 2019 (COVID-19) intensive care unit (ICU) patients. Neutrophils and PBMCs were separated and stimulated with the mitogen phytohemagglutinin. Culture supernatants of mitogen-stimulated PBMCs and neutrophils from 88 COVID-19 ICU patients and 88 healthy controls were evaluated for levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, interleukin (IL)-2, -4, -5, -6, -9, -10, -12, -17A, and tumor necrosis factor (TNF)-α using anti-cytokine antibody MACSPlex capture beads. Cytokine profiles of PBMCs showed significantly lower levels of GM-CSF, IFN-γ, IL-6, IL-9, IL-10, IL-17A, and TNF-α (p < 0.0001) in COVID-19 ICU patients. In contrast, COVID-19 ICU patients showed higher median levels of IL-2 (p < 0.001) and IL-5 (p < 0.01) by PBMCs. As for neutrophils, COVID-19 ICU patients showed significantly lower levels of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-17A, IL-12, TNF-α (p < 0.0001), and IFN-α (p < 0.01). T-helper (Th)1:Th2 cytokine ratios revealed lower inflammatory cytokine for PBMCs and neutrophils in COVID-19 ICU patients. Cytokine production profiles and Th1:Th2 cytokine ratios suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has an immunomodulatory effect on PBMCs and neutrophils. This study also suggests that the increased levels of several cytokines in the serum are not sourced from PBMCs and neutrophils.

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Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus

Cited by 13 publications

References 17 publications

Development of an optimized colorimetric RT-LAMP for SARS-CoV-2 assay with enhanced procedure controls for remote diagnostics

Development of an optimized colorimetric RT-LAMP for SARS-CoV-2 assay with enhanced procedure controls for remote diagnostics

Serological responses to the BNT-162b2 Pfizer/BioNTech vaccination in twin pregnancies- Comparison between immunization before conception and during pregnancy.

Influence of SARS-COV-2 Infection on Cytokine Production by Mitogen-Stimulated Peripheral Blood Mononuclear Cells and Neutrophils in COVID-19 Intensive Care Unit Patients

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