[SEM] QUIZ 3 - HS SARS-CoV-2 detection using graphene oxide-multiplex qPCR

Reverse transcription-quantitative polymerase chain reaction

What is the gold standard for SARS-CoV-2 detection?

Low-viral loads

rt-qpcr has only been able to detect what?

single-stranded DNA

what can graphene oxide absorb?

Graphene Oxide

what absorbs ssDNA and has been widely applied in molecular pathogen detection?

RdRp

E

what two SARS-CoV-2 genes can GO-multiplex qPCR detect?

RNase P

which reference gene can the GO-multiplex qPCR detect?

10 copies/reaction

GO-multiplex qPCR reduces the limit of detection by 10-fold to what?

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

What virus has caused the coronavirus disease in 2019?

600 million confirmed cases

more than 6 million deaths

As of August 2022, what are the statistics of the virus?

respiratory tract

The pathogen is mainly transmitted through?

accurate detection

what is one of the basic requirements for screening COVID-19 infections?

71%

conventional RT-qPCR only detects _______ of SARS-CoV-2019 infections.

insufficient assay sensitivity

low sample concentrations caused by low-viral-load in vivo

improper clinical sampling

reasons as to why RT-qPCR only detects 71% of COVID infections

Multiplex qPCR

what is the commonly adopted assay for gene expression and single nucleotide polymorphism (SNP) analysis?

Multiplex qPCR

what detects different target genes simultaneously?

complexity of the system

possible interference between different amplification reactions

reasons as to why the application of multiplex qpcr in clinical diagnosis is low?

tuberculosis

ovarian cancer

graphene oxide enhances the sensitivity of conventional PCR and single qPCR for detecting which diseases?

graphene oxide

is an oxidation derivative of graphene

hexagonal honeycomb structure

the sp2 region of GO has a structure of?

oxygen-containing functional groups

the sp3 carbon matrix region of GO is linked to?

aromatic rings

fluorescence quenching ability

the sp2 structural domain gives GO an affinity for?

hydrophilic group

the _____ attached to the sp3 region stabilizes GO in aqueous solutions

large surface area

outstanding chemical functional groups

unique interfacial properties

three properties of GO that gives it a strong ability to adsorb ssDNA

pi stacking

hydrogen bonding

GO adsorbs ssDNA through?

Hummers’ method

this is the method used by the researchers to prepare GO

sp2 to sp3 ratio

the synthesis involved adjusting the GO oxidative degree to an appropriate ______?

forward primers of the target genes

what was absorbed by GO to construct the GO-forward primer composites?

fluorescence quenching

shift in the UV absorption peak

these two confirmed the formation of GO-forward primer composites

go-forward primer composites

what significantly improves the sensitivity of multiplex qRT-PCR?

Qing-dao Laixi Colloidal Graphite Factory

Graphite was purchased where?

Potassium Permanganate

Potassium Nitrate

Sulfuric Acid

30% hydrogen peroxide

What were obtained from Chengdu Kelong Chemical Reagent Factory?

Shanghai Yuanye Biological Science and Technology Company

The 3500 Da dialysis bag was purchased from?

ultrapure water

this was used to prepare all aqueous solutions

25 degrees C

temperature of the ultrapure water used for aqueous solutions

95%-98%

percentage of sulfuric acid used for graphene oxide stock solution

0.5g

how many grams of carbon powder were used for the GO stock solution?

3g of KMnO4

0.5g of NaNO3

what were added to the GO stock solution an stirred well to form dark green MnO3?

dark green

color of MnO3 once KMnO4 and NaNO3 were added to the solution?

H2O2 (2.5 mL or 10 mL)

what was added to the embedded graphene for oxidation to form the GO stock solution?

dialysis in deionized water

the residual acid and low molecular weight reaction products in the GO solution where eliminated through?

the FEI Tecnai G2 F30 S-TWIN transmission electron microscope

this was used for transmission electron microscopy

Reinshaw inVia microscope

Raman scattering was performed using which microscope?

Fourier transform infrared spectroscopy technique

the infrared spectra were obtained using which technique?

NICOLET IS10 FT-IR spectrometer

the infrared spectra were obtained using which equipment?

MAPADA UV6300 UV-visible spectrophotometer

which was used to measure the ultraviolet-visible (UV-vis) absorption trophotometer

Thermo ESCALAB 250XI scanning XPS microscope

the x-ray photoelectron spectroscopy pattern of the samples was measured by?

WHO

CDC

the primers and probes for RdRP, E, and RNase P genes were design based on the methods of?

4’,5’-dichloro-2’,7’-dimethoxy-6-carboxyfluorescein

the specific probe for RdRP was labeled with?

carboxyrhodamine

the specific probe for E was labeled with?

hexachlorofluorescein

the specific probe for RNase P was labeled with?

Sangon Biotech

this company synthesized all the primers and probes

20 minutes

the GO stock solution was pre-sonicated for how many minutes?

one-to-one ratio

ratio of the GO dilution mixed with 10 uM forward primers of RdRP, E and RNase P.

RNase-free ddH2O

was used as a control group after processing at room temperature

12.5 uL of Perfect StartTM II probe qPCR SuperMix

the “base”

0.5 uL each of 10uM

measurements of each primer

1 uL

measurement of GO-forward primers

0.5 uL

measurement of regular forward primer

3.5 uL

RNase-free water measurement for GO-multiplex

5uL

RNase-free water measurement for conventional PCR

40 cycles

number of cycles done in both GO-multiplex and conventional PCR

<=37

Ct value considered positive

coronavirus HKU1

human parainfluenza virus

RNA viruses used for the specificity of the GO-multiplex

Enterovirus

Adenovirus

Rhinovirus

Human cytomegalovirus

Varicella-zoster virus

Mumps

Measles

7 DNA viruses used for the specificity of GO-multiplex