Diagnostic tech

Question 1

What is the primary benefit of Next Generation Sequencing (NGS) over traditional methods for pathogen detection?

Answer 1

NGS provides a comprehensive analysis that can discover novel or unexpected pathogens without prior knowledge of their genetic sequence, unlike targeted methods. It can also identify multiple organisms simultaneously using metabarcoding.

Question 2

Explain the principle behind Isothermal Amplification.

Answer 2

Isothermal amplification methods like LAMP and RPA amplify specific nucleic acid sequences at a constant temperature, which eliminates the need for a thermal cycler. This makes them highly suitable for field-deployable diagnostics.

Question 3

How does a CRISPR-based diagnostic system like SHERLOCK generate a detectable signal?

Answer 3

A CRISPR enzyme (e.g., Cas12 or Cas13) is programmed with a guide RNA to bind to a specific target sequence. Upon binding, the enzyme activates a collateral cleavage activity that cuts reporter molecules, generating a fluorescent or visual signal.

Question 4

What is the significance of the Quantification Cycle (Cq) in Quantitative PCR (qPCR)?

Answer 4

The Cq value is the cycle number at which the fluorescence from an amplifying target crosses a defined threshold. It is inversely related to the initial amount of target nucleic acid in the sample, allowing for quantification of the pathogen.

Question 5

What is the main advantage of Digital PCR (dPCR) over qPCR?

Answer 5

dPCR provides absolute quantification of the target DNA/RNA, meaning it measures the exact number of copies without needing a standard curve. It is also more tolerant of PCR inhibitors.

Question 6

How does an ELISA detect a pathogen, and what does it primarily target?

Answer 6

ELISA uses an antibody to bind to a specific antigen (usually a pathogen protein). An enzyme linked to a secondary antibody produces a measurable signal (e.g., a color change) upon reaction with a substrate, confirming the presence of the antigen.

Question 7

What is the key benefit of Lateral Flow Assays (LFAs) for field use?

Answer 7

LFAs are simple, rapid, inexpensive, and portable, requiring no power and providing a result in minutes, making them ideal for point-of-care use in the field.

Question 8

Why is Hyperspectral Imaging useful for early disease detection in plants?

Answer 8

Hyperspectral imaging can detect subtle physiological changes in plants caused by stress or disease that alter how they reflect/absorb light. These changes often occur before visible symptoms are present.

Question 9

What is a major limitation of NGS related to data analysis?

Answer 9

A significant limitation is the expertise required for data analysis and interpretation, as NGS generates large, complex datasets.

Question 10

Why is the primer design for Isothermal Amplification methods like LAMP considered complex?

Answer 10

Primer design is complex because LAMP uses 4-6 primers that must recognize and bind to multiple distinct sequence regions for the amplification to work efficiently.

Question 11

What is the primary benefit of CRISPR-based diagnostics over other molecular methods?

Answer 11

CRISPR-based diagnostics have exceptional specificity, capable of distinguishing even single nucleotide differences between pathogens.

Question 12

Why is qPCR considered a quantitative method?

Answer 12

It accurately measures the initial amount of nucleic acid in a sample by monitoring the fluorescence intensity in real-time, which is proportional to the amount of DNA being amplified.

Question 13

How does dPCR perform absolute quantification without a standard curve?

Answer 13

It partitions a reaction into thousands of individual reactions (wells or droplets) and counts the number of positive reactions. This is then used to calculate the absolute number of copies in the original sample based on Poisson statistics.

Question 14

What is the main limitation of an ELISA compared to a rapid field test like LFA?

Answer 14

ELISA typically requires a controlled laboratory setting with specialized equipment, whereas an LFA can be used on-site without any equipment.

Question 15

What is a drawback of using Lateral Flow Assays (LFAs) for pathogen detection?

Answer 15

LFAs are generally less sensitive than molecular methods and may miss low levels of a pathogen. They are also qualitative, providing a yes/no result rather than a quantification.

Question 16

What is a major benefit of using Portable PCR & LAMP/RPA devices?

Answer 16

They provide the power of sensitive and specific on-site molecular testing, which gives rapid and accurate results without the need to transport samples to a lab.

Question 17

What is the difference between Hyperspectral and Multispectral imaging?

Answer 17

Hyperspectral imaging captures images across many narrow wavelengths, while multispectral imaging captures images across fewer, broader wavelengths.

Question 18

What is the major limitation of molecular diagnostics (NGS, LAMP, qPCR, etc.) regarding pathogen viability?

Answer 18

These techniques detect the pathogen’s genetic material (DNA/RNA) but do not confirm whether the pathogen is still living and infectious.

Question 19

What does metabarcoding allow for in the context of NGS?

Answer 19

Metabarcoding allows for the simultaneous identification of many organisms within a single sample, providing a comprehensive view of the microbial community.

Question 20

What is a key benefit of Isothermal Amplification methods compared to PCR regarding sample preparation?

Answer 20

They are often more robust and tolerant to inhibitors found in unpurified plant samples, which can simplify the sample preparation process.