Deck 8: Nucleic Acid-Based Analytic Methods for Microbial Identification and Characterization

Question 1

The optimal way to detect PCR amplicons is to use which stain?

A)Methylene blue
B)Malachite green
C)Bromothymol blue
D)Ethidium bromide

Answer 1

D
The nucleic acid bands in the agarose gel are stained with the fluorescent dye,ethidium bromide,which allows them to be visualized on exposure to ultraviolet (UV)light.

Question 2

Nucleic acid sequencing methods are based on:

A)the ability of two nucleic acid strands that have complementary base sequences to bond specifically with each other and form a double-stranded molecule.
B)detecting phenotypic traits not detectable by conventional strategies.
C)the principles of complementary nucleic acid hybridization with those of nucleic acid replication.
D)determining the exact nucleotide sequence of a gene or gene fragment obtained from an organism.

Answer 2

D
The nucleotide sequence of a microorganism's genome is the blueprint for that organism;therefore,the knowledge of its nature is a valuable tool in identifying infectious agents in clinical specimens.

Question 3

Hybridization methods are based on:

A)the ability of two nucleic acid strands that have complementary base sequences to bond specifically with each other and form a double-stranded molecule.
B)detecting phenotypic traits not detectable by conventional strategies.
C)the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are repeatedly applied through numerous cycles.
D)determining the exact nucleotide sequence of a gene or gene fragment obtained from an organism.

Answer 3

A
The ability of complementary nucleic acid strands to bond specifically with each other and form a double-stranded molecule is what drives hybridization methods.

Question 4

Gen-Probe Mycobacterium tuberculosis direct test uses which type of nucleic acid amplification test?

A)Nucleic acid sequence-based amplification (NASBA)
B)Transcription-mediated amplification (TMA)
C)Reverse transcriptase polymerase chain reaction (RT-PCR)
D)Standard displacement amplification (SDA)

Question 5

An organism whose genetic makeup is 50% C-G bonds would have a(n)_________ melting temperature (Tm),compared with an organism with 20% C-G bonds.

A)higher
B)lower
C)equal
D)similar

Question 6

Which of the following steps are crucial for the success of a nucleic acid amplification test,such as PCR?

A)Nucleic acid purification
B)Fluorescent resonance energy transfer (FRET)
C)Melting temperature analysis
D)Nucleic acid sequencing

Question 7

After PCR,the amplification control has failed to yield a product.What should you do?

A)Report out Indeterminate
B)Report out Negative because there was no amplification from the sample
C)Check the original DNA or RNA preparation.If it is adequate,repeat the amplification.If not,re-isolate the nucleic acid.
D)Repeat it using a higher concentration of amplification control in the reaction.

Question 8

The enzyme that has the ability to synthesize DNA from ribonucleic acid (RNA)is called:

A)DNA polymerase.
B)RNAse.
C)helicase.
D)reverse transcriptase.

Question 9

The target in the hybridization assay can be described as:

A)originating from the unknown organism to be detected or identified.
B)a reporter molecule that chemically forms a complex with the single-stranded probe DNA.
C)originating from an organism of known identity.
D)an enzyme that adds nucleotides during the replication process.

Question 10

Which type of hybridization method would be the optimal choice to use to detect a lesion infected with a particular virus?

A)Southern
B)In situ
C)Peptide nucleic acid
D)Sandwich

Question 11

Determining the exact nucleotide sequence of a gene or gene fragment obtained from an unknown organism is based on which of the following methods?

A)Hybridization
B)Sequencing
C)Amplification (PCR)
D)Genotypic

Question 12

Analysis of the number of target molecules in the original clinical specimen can be determined using which of the following molecular techniques?

A)Arbritary Primed PCR
B)Peptide Nucleic Acid (PNA)FISH
C)Nested PCR
D)Quantitative PCR

Question 13

Which enzyme recognizes a specific nucleotide sequence (~4 to 8 nucleotides in length)and catalyzes the digestion of the nucleic acid at that site,causing a break in the DNA strand?

A)Ligase
B)Gyrase
C)Helicase
D)Endonuclease

Question 14

The ability of two nucleic acid strands,a probe and a target,which have complementary base sequences that specifically bond with each other and form a double-stranded molecule,drives which of the following methods?

A)Hybridization
B)Sequencing
C)Amplification (PCR)
D)Genotypic

Question 15

The enzyme that is commonly used for primer extensions in the PCR method works best at what temperature?

A)25°C
B)56°C
C)72°C
D)94°C

Question 16

Complementary nucleic acid hybridization,which is coupled with nucleic acid replication and applied repeatedly through numerous cycles,is based on which of the following methods?

A)Hybridization
B)Sequencing
C)Amplification (PCR)
D)Genotypic

Question 17

The probe in the hybridization assay can be described as:

A)originating from the unknown organism to be detected or identified.
B)a reporter molecule that chemically forms a complex with the single-stranded probe deoxyribonucleic acid (DNA).
C)originating from an organism of known identity with a known sequence.
D)the enzyme needed to elongate the strand.

Question 18

Target nucleic acid amplification (polymerase chain reaction [PCR])methods are based on:

A)the ability of two nucleic acid strands that have complementary base sequences to bond specifically with each other and form a double-stranded molecule.
B)detecting phenotypic traits not detectable by conventional strategies.
C)the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are repeatedly applied through numerous cycles.
D)determining the exact nucleotide sequence of a gene or gene fragment obtained from an organism.

Question 19

Analytical sensitivity is concerned primarily with which of the following PCR parameters?

A)Target specificity
B)Limit of detection
C)Head-to-head comparison to a gold-standard test
D)Gram staining

Question 20

Predict which of the following DNA oligonucleotide probes would have the highest melting temperature (Tm)when hybridizing to the target sequence 5'-GTGCCTGAATGCAATGTCA-3'.

A)3'-CACGGA-5'
B)3'-CATGGA-5'
C)3'-TTACAG-5'
D)3'-TTACTG-5'

Question 21

Which of the following molecular techniques,or combination of techniques,would be most appropriate for detection and quantitation of an RNA retrovirus,like HIV,in a blood sample in order to establish the patient's viral load?

A)RT-PCR
B)Real-time PCR
C)Real-time RT-PCR
D)Multiplex PCR

Question 22

While developing a new hybridization-based molecular test for Cryptosporidium,a protozoan parasite that causes diarrheal illness,a laboratory technician examines the hybridization ability with a closely related,but nontarget,protozoan and also gets a positive test result.In order to improve the stringency of the hybridization reaction for detection of Cryptosporidium only,the technician can do which of the following?

A)Decrease the salt concentration in the hybridization buffer
B)Increase the salt concentration in the hybridization buffer
C)Reduce the reaction temperature during hybridization
D)Remove destabilizing agents,such as formamide,from the hybridization buffer
E)None of the above

Question 23

Identify the appropriate DNA homolog,or complementary sequence,that could be used as a probe for in situ hybridization to the following target RNA: 5'-AUGCGAUUC-3'

A)5'-TACGCTAAG-3'
B)5'-ATGCGATTC-3'
C)5'-GAATCGCAT-3'
D)5'-AUGCGAUUC-3'

Question 24

If a patient sample contains 8 copies of viral DNA,predict how many DNA copies would be present following three rounds of PCR amplification.

A)16
B)24
C)32
D)64

Question 25

Isothermal amplification techniques,such as NASBA,utilize a single temperature for nucleic acid amplification thereby eliminating the need for thermal cycling instruments to precisely and quickly alter the reaction temperature.This often reduces the cost associated with molecular testing and infrastructure requirements.In what settings could this be advantageous?

A)Developing countries with limited health care facilities
B)Field clinics during military operations
C)Small clinics or hospitals without centralized laboratory facilities
D)Urgent care clinics or pharmacies for point-of-care diagnosis
E)All of the above

Question 26

Gel electrophoresis is used to separate nucleic acid amplicons based upon what physical property?

A)Sequence
B)Duplex formation
C)Size
D)Secondary folding structure

Question 27

What key advantage do emerging technologies,such as MALDI-TOF and next generation sequencing,have over conventional hybridization and amplification techniques?

A)They do not require selection of suspected pathogens prior to testing.
B)They do not require expensive instrumentation or skilled technicians.
C)They eliminate sample preparation steps,such as nucleic acid purification,prior to testing.
D)They are readily available in most clinical laboratories.
E)All of the above.

Question 28

Place the basic steps involved in a molecular hybridization assay in sequence from first to last in the order in which they would be performed.

A)Heating sample to obtain single-stranded target nucleic acid,hybridization of target to probe,detection of hybridization,labeling of probe
B)Labeling of probe,heating sample to obtain single-stranded target nucleic acid,hybridization of target to probe,detection of hybridization
C)Hybridization of target to probe,detection of hybridization,labeling of probe,heating sample to obtain single-stranded target nucleic acid
D)Hybridization of target to probe,labeling of probe,heating sample to obtain single-stranded target nucleic acid,detection of hybridization

Question 29

What differentiates diagnostic techniques that employ phenotypic (culture-based)methods from those that employ molecular methods?

A)Phenotypic methods are highly sensitive and specific,while molecular methods are not.
B)Phenotypic methods examine the growth behavior of a microorganism and the proteins,enzymes,or toxins produced,while molecular methods examine the genetic makeup.
C)Phenotypic methods are quantitative while molecular methods are not.
D)Phenotypic methods are not limited to detection of live organisms as are molecular methods.