Test 2- Diagnosis of Viral Techniques

Question 1

Polymerase chain reaction (PCR)

Answer 1

 Polymerase chain reaction, or PCR, is a laboratory technique used to make multiple copies of a segment of DNA.

 PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules.

 Sometimes called “molecular photocopying,” the polymerase chain reaction (PCR) is a fast and inexpensive technique used to “amplify” - copy - small segments of DNA.

 The amplified DNA is subjected to electrophoresis in agarose gels and can be visualized by ethidium bromide staining.

BAND= VIRUS BEING THE SAMPLE

Question 2

Real-Time Polymerase chain reaction (PCR)

Answer 2

 Real-Time PCR or Quantitative PCR is an advanced form of PCR which allows monitoring and quantification of increasing accumulation of PCR products/nucleic acid load as the reaction progresses. Useful to study virus load in patient.

 In real-time PCR assays:
(1) A target specific probe (labelled with a fluorescent dye), such as the 5’-nuclease TaqMan

probes, molecular beacons, or FRET hybridization probes,
(2) Or intercalating dyes, such as SYBR Green
are used in an otherwise conventional PCR reaction. The fluorescence emitted by the excited fluorophore (Fluorescent probes or dye) is visualized and analyzed.

Question 3

Genome sequencing

Answer 3

 DNA sequencing refers to the process by which the sequence of bases in a DNA molecule is elucidated/can be obtained and read.

 The most widely used method of DNA sequencing is the modification of the Sanger dideoxy method, which involves using chain-terminating dideoxynucleotides.

traces origin, transmission of a virus

Question 4

Next Generation Sequencing

Answer 4

 Currently, surveillance studies are more relying on Next Generation Sequencing (NGS) technologies that are gradually replacing the conventional sequencing methods.

 NGS platforms, such as Illumina (Solexa) sequencing, are significantly cheaper, quicker, needs significantly less DNA, has high throughput, and is more accurate and reliable than Sanger sequencing.

Question 5

Metagenomics

Answer 5

 Metagenomics: Metagenomics is defined as the study of the collective set of microbial populations in a sample by analyzing the sample’s entire nucleotide sequence content, and is a powerful method for random detection of existing and new pathogens.

 This is achieved by amplification and sequencing of whole genome (DNA and/or RNA) content of a given sample followed by filtering and analysis of obtained data by comparing with genome databases and using different softwares.

 Next Generation Sequencing Platforms (NGS platforms), owning to their high- throughput sequencing capacity, are being extensively used in this type of studies.

Question 6

 Genome sequencing plays a crucial role in surveillance studies, as it allows:

Answer 6

 Genome sequencing plays a crucial role in surveillance studies, as it allows:

(1) Pathogen detection
(2) Studies on genetic variation, such as genotyping, evolution and interspecies transmission of pathogens

(3) Identification of novel and undiscovered strains
(4) Development of diagnostics, such as genotyping primers, or probes
(5) Identification of genes associated with drug resistance
(6) Development of therapeutics
(7) Judging the efficacy of current vaccines and formulating new vaccine strategies

Question 7

Phylogenetic Analysis

Answer 7

Phylogenetic Analysis: The use of Virus genome sequence data to study evolution of viruses and genetic relationships among viruses

Question 8

Microarrays

Answer 8

 In microarray, several thousands of known DNAs (probes), amplified by PCRs/RT-PCRs, are spotted onto a glass or a silicon chip.

 The target/sample DNA are fluorescently labeled and then hybridized/added to the chip containing DNA probes.

 Positive reactions between probe-DNA and Sample DNA (hybridization) generate a fluorescent signal from the spot where probe DNA is spotted in the chip.

 The intensity of fluorescence can be measured using a fluorescent scanner, and the data analyzed by a variety of methods.

 The advantage of microarrays in detection of pathogens in surveillance studies is that hundreds of pathogens can be screened for simultaneously using a single microarray chip.

You can screen for hundreds of pathogens in a single chip

Question 9

Steps in a PCR

Answer 9

1. Denaturation: To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA.
2. Annealing: Two short stretches of nucleotides (20-30 nucleotides long), known as primers, attach to ends of each of the two separated DNA segments. Forward primer (+ sense) binds to reverse DNA strand (- sense strand) and vice versa.
3. Extension/Elongation: Next, an enzyme called “Taq polymerase” synthesizes - builds -two new strands of DNA using the original strands as templates . Taq Polymerase keeps on adding free nucleotides (complementary to the corresponding nucleotides of original DNA segment) to the attached primer. Thus, a new DNA segment extending from the primer, and complementary to original DNA segment is created.