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Questions and Answers

What is primarily indicated by an R² value of 0.998 in a qPCR assay?

There is a complete lack of specificity in the assay.

There is a strong correlation between Cq value and the amount of target DNA. (correct)

The model does not explain the variation in the data.

The assay is poorly optimized for sensitivity.

Which step is crucial in the development of custom qPCR assays?

Conducting validation and optimization studies. (correct)

Only using commercially available assays.

Using a universal primer design for all assays.

Skipping validation to save time.

When discussing amplicon design, what should researchers aim for to ensure reliable results?

Primers that show strong cross-dimerization potential.

Minimizing the homology of target sequences.

Using low-fidelity DNA polymerases.

High-quality primers with known hairpin structures. (correct)

What benefit does custom assay development provide over standard commercially available assays?

They are specifically suited to particular research goals. Signup and view all the answers

What is the potential drawback of using assays without validation?

They could lead to unreliable data and conclusions. Signup and view all the answers

In sensitivity analysis, what does a high temperature tolerance indicate?

Greater ability to function under varied conditions. Signup and view all the answers

What role does resource synthesis have in assay development?

It can facilitate the creation of novel test methods. Signup and view all the answers

What is a primary concern when selecting primers for amplification?

The potential for high cross-dimerization. Signup and view all the answers

What does a slope of -3.32 in a PCR assay indicate?

The assay is 100% efficient Signup and view all the answers

How many replicates are needed for an acceptable evaluation of PCR efficiency?

Four to five replicates Signup and view all the answers

What role does a linear standard curve with an R2 value greater than 0.980 serve in PCR?

It shows how efficient the PCR reaction is Signup and view all the answers

What is essential for achieving high amplification efficiency in PCR as indicated in the content?

Avoiding any primer dimers Signup and view all the answers

What can be inferred about an assay's sensitivity with lower efficiency?

It will result in higher Cq values Signup and view all the answers

What parameter indicates that there are no secondary structure issues at the primer binding sites?

A gradient recording similar q values Signup and view all the answers

What does the presence of primer dimers affect in a PCR assay?

It decreases the standard curve accuracy Signup and view all the answers

Which element is critical for reliable qPCR according to the information provided?

Good primers design Signup and view all the answers

What is the primary reason to prevent short amplicons in qPCR?

To differentiate between amplicon and primer dimer. Signup and view all the answers

What does a high amplification efficiency in qPCR typically range between?

95% - 105% Signup and view all the answers

What aspect allows quantification in qPCR assays?

A linear relationship between initial template quantity and Cq value. Signup and view all the answers

Which of the following describes a hallmark of an optimized qPCR?

Consistency across replicate experiments. Signup and view all the answers

How is the dynamic range of a well-designed qPCR assay best characterized?

By a linear relationship between Cq value and template quantity. Signup and view all the answers

What is a common misconception about the relationship between Cq value and assay sensitivity?

Cq value is directly tied to a specific copy number. Signup and view all the answers

Which method is used to generate a standard curve in qPCR?

Using serial dilutions of a template. Signup and view all the answers

Why is consistency across replicate experiments important in qPCR?

It ensures reliability of results across different labs. Signup and view all the answers

Study Notes

High sensitivity SARS-CoV-2 detection using graphene oxide-multiplex qPCR

RT-qPCR is the gold standard for detecting SARS-CoV-2, but only detects 71% of infections. Insufficient assay sensitivity and low sample concentrations can occur due to low viral load or improper sampling.
Multiplex qPCR is commonly used for gene expression and SNP analysis. It detects multiple target genes simultaneously, but is complex and can be interfered with by other amplification reactions.
Graphene oxide (GO) is an oxidation derivative of graphene. It has sp² regions with a hexagonal honeycomb structure, and sp³ carbon matrix regions linked to oxygen-containing functional groups. The sp² region gives GO an affinity for aromatic rings and fluorescence-quenching ability. The hydrophilic groups attached to the sp³ region stabilize GO in aqueous solutions.

Multiplex qPCR using graphene oxide

GO-multiplex qPCR significantly improves SARS-CoV-2 detection, especially for low-viral-load samples.
This method reduces the detection limit by 10-fold (to 10 copies/reaction).
GO reduces non-specific primer interactions, enhancing specificity and preventing cross-reactivity with other respiratory viruses.
GO-multiplex qPCR shows better amplification efficiency and lower cycle threshold (Ct) values compared to conventional methods.
The study focused on developing a qPCR method using GO-multiplex for SARS-CoV-2 detection.
Nine common respiratory RNA and DNA viruses were used for specificity tests regarding cross-reactivity.

Optimization of GO-multiplex qPCR

Optimization of GO solutions involved varying H₂O₂ volumes (2.5mL and 10mL). GO prepared with 2.5mL H₂O₂ demonstrated better Ct values for RdRP, E, and RNase P genes, potentially due to a higher sp² region ratio and larger surface area.
Optimization of the temperature for GO-forward primer composites found 50°C to be the optimum temperature to maximize GO adsorption to primers, maximizing fluorescence quenching efficiency.
GO-multiplex qPCR exhibited improved sensitivity with a detection limit of 10 copies per reaction, notably lower than conventional multiplex qPCR.

Assay Validation

The GO-multiplex qPCR method was validated using SARS-CoV-2 pseudoviruses with RdRP and E target sequences.
The results of this validation, comparing GO-multiplex qPCR to conventional multiplex qPCR, showed that GO-multiplex qPCR effectively detected SARS-CoV-2 at very low viral loads.