Question 1

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

Accepted Answer

The combination of real-time,or quantitative,detection methods with RT-PCR would allow for quantitative determination of a viral RNA transcript.

Question 2

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

Accepted Answer

Stringency in a hybridization assay is affected by the composition of the hybridization buffer,with higher salt concentrations and the presence of destabilizing agents increasing stringency,thereby reducing nontarget hybridizations.In addition,higher temperatures of the hybridization reaction improve stringency by ensuring a higher degree of complementarity between the probe and target nucleic acids,reducing tolerance for nucleotide mismatches that may be present in a closely related,but nontarget sequence.

Question 3

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'

Accepted Answer

Duplex formation is driven by the hydrophobic structure and hydrogen bonding pattern of the nucleotides,which ensure that,in DNA,the base adenine always bonds to thymine and guanine always bonds to cytosine.In RNA,the same base pairing rules follow for guanine and cytosine,but uracil replaces thymine to form a base pair with adenine.In addition,the two strands run antiparallel,5' to 3' on one strand and 3' to 5' on the other.Due to convention,sequences are typically written in the 5'-3' direction.

Question 4

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

Accepted Answer

In amplification PCR-based testing,each cycle in the reaction doubles the amount of nucleic acid present and each new copy serves as a template for the next round,yielding an exponential amplification in copy number (2ⁿ).

Question 5

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

Accepted Answer

Isothermal techniques are well suited for any location or setting where resources (technical personnel,expensive instrumentation,electrical power,etc. )are limited or when diagnosis is needed at the point-of-care.

Question 6

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

Accepted Answer

Gel electrophoresis of amplified DNA is based upon size,or length,of the amplified nucleic acid.

Question 7

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.

Accepted Answer

MALDI-TOF and NGS allow examination of all proteins/nucleic acids within a sample and do not require identification of suspected pathogens as do hybridization/amplification techniques in order to select appropriate probe sequences.

Question 8

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

Accepted Answer

The basic steps in a hybridization assay include:
1.Production and labeling of single-strand nucleic acid probe
2.Preparation of single-strand target nucleic acid
3.Mixture and hybridization of target and probe nucleic acid
4.Detection of hybridization

Question 9

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.

Accepted Answer

Molecular methods involve the detection and manipulation of nucleic acids (deoxyribonucleic acid [DNA] and ribonucleic acid [RNA])allowing microbial genes to be examined directly (i.e. ,genotypic methods)rather than by analysis of their products,such as enzymes,other proteins and toxins,or identifiable characteristics of organism growth (i.e. ,phenotypic methods).

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

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?

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'

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

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

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

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.

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