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Molecular Biology Techniques & HIV Diagnostics

Tier 4 · #31 of 33 · lower yield · topic 2.5 — every high-yield fact from the book chapter, exam-ready.

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  1. Front
    What type of genetic material does HIV store its genome as?
    Back
    RNA (it is a retrovirus)
  2. Front
    Which host cell does HIV target and destroy?
    Back
    CD4+ T lymphocytes
  3. Front
    Which viral surface protein docks onto the CD4 receptor during HIV binding (determining viral tropism)?
    Back
    gp120
  4. Front
    Which HIV protein mediates fusion of the viral envelope with the host cell membrane?
    Back
    gp41
  5. Front
    Which HIV enzyme converts the single-stranded RNA genome into double-stranded DNA ?
    Back
    Reverse transcriptase
  6. Front
    Which HIV enzyme permanently inserts the viral DNA into the host's chromosomal DNA?
    Back
    Integrase
  7. Front
    Which HIV enzyme cleaves large precursor polyproteins into individual functional units during maturation?
    Back
    Protease
  8. Front
    Which HIV capsid protein encases the RNA genome and appears in plasma very early in infection as a diagnostic marker?
    Back
    p24
  9. Front
    Cloze: In PCR, the heat-stable DNA polymerase isolated from Thermus aquaticus is {{Taq polymerase}}.
    Back
    Taq polymerase
  10. Front
    What cofactor is required as a divalent cation for polymerase activity in PCR?
    Back
    Magnesium ( magnesium chloride / Mg2+ )
  11. Front
    What is the function of the two primers in PCR?
    Back
    Short single-stranded oligonucleotides that flank (and define the boundaries of) the region to be amplified
  12. Front
    What happens during the denaturation step of PCR, and at what temperature?
    Back
    Double-stranded template separates into single strands at 95°C
  13. Front
    What happens during the annealing step of PCR, and at roughly what temperature?
    Back
    Primers bind to their complementary sequences at ~55°C
  14. Front
    At what temperature does Taq polymerase extend the new strand during the PCR extension step?
    Back
    72°C
  15. Front
    After n PCR cycles, the number of target DNA copies is given by what formula?
    Back
    copies = 2^n
  16. Front
    A single starting DNA molecule becomes roughly how many copies after 30 PCR cycles?
    Back
    Over one billion (~10^9) copies
  17. Front
    Why does PCR ( Taq polymerase ) introduce errors at a low rate?
    Back
    Taq polymerase has no proofreading (3'→5' exonuclease) ability
  18. Front
    What problem does RT-PCR solve compared to standard PCR?
    Back
    It allows amplification when the starting template is RNA , by first converting RNA to cDNA
  19. Front
    In RT-PCR , what product does reverse transcriptase generate from the RNA template?
    Back
    Complementary DNA (cDNA)
  20. Front
    Which primer is used in RT-PCR to convert only mRNA (not rRNA or tRNA) into cDNA ?
    Back
    Oligo-dT primer (a short chain of 12–18 deoxythymidine residues)
  21. Front
    Why does the oligo-dT primer specifically reverse-transcribe mRNA and not rRNA or tRNA?
    Back
    Oligo-dT anneals to the poly(A) tail at the 3' end of eukaryotic mRNA ; rRNA and tRNA lack a poly(A) tail
  22. Front
    Why is cDNA made from mRNA free of introns?
    Back
    It is copied from processed (spliced) mRNA , which has already had introns removed
  23. Front
    In RT-PCR expression analysis, why are primers chosen to span an exon-exon junction ?
    Back
    So genomic DNA contamination yields a larger (intron-containing) product distinguishable by size from true cDNA
  24. Front
    In RT-qPCR , what fluorescent reporter dye binds double-stranded DNA and increases signal as product accumulates?
    Back
    SYBR Green
  25. Front
    What is the threshold cycle (Ct) in RT-qPCR ?
    Back
    The cycle number at which the fluorescence signal first crosses a set threshold above baseline
  26. Front
    What is the relationship between Ct value and the starting amount of target template?
    Back
    Inverse : low Ct = more starting template (high viral load) ; high Ct = less template
  27. Front
    Two HIV samples give Ct = 18 and Ct = 32 . Which has the higher viral load?
    Back
    Ct = 18 ( lower Ct = more starting RNA = higher viral load )
  28. Front
    Which phase of the RT-qPCR amplification plot is the most analytically reliable?
    Back
    The exponential phase (product doubles each cycle)
  29. Front
    Why does an RT-qPCR reaction eventually reach a plateau ?
    Back
    One or more reaction components (reagents) become exhausted
  30. Front
    How are Ct values converted into absolute copy numbers in RT-qPCR ?
    Back
    By interpolating from a standard curve (Ct vs. log starting quantity, built from known dilutions)
  31. Front
    What is the approximate lower detection limit of current HIV viral load testing?
    Back
    ~50 copies/mL of plasma
  32. Front
    What is the eclipse period in HIV infection?
    Back
    The time between infection and appearance of the first detectable marker, during which no test can detect infection
  33. Front
    Which marker first appears to end the HIV eclipse period , and at about what day?
    Back
    HIV RNA , detectable around day 12 (in ~50% of patients)
  34. Front
    What is the window period in HIV testing (and how does it differ from the eclipse period )?
    Back
    The time between exposure and when a SPECIFIC test can reliably detect infection; it is test-specific , whereas the eclipse period is fixed (no test detects)
  35. Front
    At about what day does p24 antigen become detectable in plasma after HIV infection?
    Back
    ~Day 15
  36. Front
    Why does p24 antigen become undetectable around day 50 of HIV infection?
    Back
    Early anti-HIV antibodies form immune complexes with p24 , clearing it from the blood
  37. Front
    Which antibody class appears FIRST in HIV infection (~day 20)?
    Back
    IgM
  38. Front
    At about what day does IgG production begin in HIV infection?
    Back
    ~Days 30–35
  39. Front
    Roughly what percentage of all blood antibodies is IgG ?
    Back
    ~75–80%
  40. Front
    Which immunoglobulin class is the only one capable of crossing the placenta?
    Back
    IgG
  41. Front
    What is the correct order of appearance of HIV markers after infection?
    Back
    HIV RNA → p24 antigen → IgM → IgG
  42. Front
    What is the standard first-line SCREENING test for HIV ?
    Back
    ELISA (EIA, Enzyme-Linked Immunosorbent Assay)
  43. Front
    In a basic HIV ELISA , what is the patient's serum being tested for?
    Back
    Anti-HIV antibodies
  44. Front
    What two markers does a 4th-generation (Ag/Ab combination) ELISA detect simultaneously?
    Back
    Anti-HIV antibodies AND p24 antigen
  45. Front
    Why can the 4th-generation ELISA detect HIV earlier than antibody-only tests (~2-week window)?
    Back
    It detects p24 antigen , which appears before antibodies are produced
  46. Front
    Which ELISA generation first detected both IgG and IgM (sandwich format), shortening the window to ~3 weeks?
    Back
    Third-generation
  47. Front
    Why is a positive ELISA result never diagnostic for HIV on its own?
    Back
    ELISA is a screening test optimized for sensitivity , so it can give false positives ; every reactive result needs a confirmatory test
  48. Front
    What is the classical CONFIRMATORY test for HIV ?
    Back
    Western blot
  49. Front
    Onto what membrane are HIV proteins transferred during a Western blot ?
    Back
    Nitrocellulose membrane
  50. Front
    How are HIV proteins separated in the first stage of a Western blot ?
    Back
    By size using gel electrophoresis (smaller proteins migrate farther)
  51. Front
    What are the three bands used in the CDC criteria for a positive HIV Western blot ?
    Back
    p24, gp41, and gp120/gp160
  52. Front
    How many of the three diagnostic bands (p24, gp41, gp120/160) are required for a POSITIVE HIV Western blot ?
    Back
    At least two of the three
  53. Front
    How is a Western blot with some bands but not meeting positive criteria (e.g. a single p24 band) classified?
    Back
    Indeterminate ( NOT negative — uninterpretable, must be followed up)
  54. Front
    Why can the Western blot NOT detect acute HIV infection?
    Back
    It depends on antibodies , which appear weeks after viral RNA
  55. Front
    In the modern HIV algorithm, what test follows a reactive 4th-generation ELISA ?
    Back
    An HIV-1/HIV-2 antibody differentiation immunoassay
  56. Front
    If a reactive ELISA is followed by an indeterminate/negative differentiation immunoassay, what test detects acute infection?
    Back
    RT-PCR for HIV-1 RNA
  57. Front
    What test is performed on every blood donation to catch acute HIV before antibodies appear?
    Back
    Nucleic acid amplification testing (NAT) for HIV-1 and HIV-2
  58. Front
    Name three causes of a FALSE POSITIVE HIV ELISA result.
    Back
    Autoantibodies (autoimmune disease) , acute infection ( EBV/HSV/syphilis ), pregnancy, recent vaccination (within 1 month), or organ transplant (any three)
  59. Front
    Name causes of a FALSE NEGATIVE HIV antibody test.
    Back
    Testing in the window period , immunodeficiency/ advanced AIDS , hypo- or agammaglobulinemia , or incorrect test performance
  60. Front
    Why can antibody-based HIV tests be falsely negative in late-stage AIDS despite active viremia?
    Back
    The depleted immune system may be unable to mount a measurable antibody response (so viral load by RT-qPCR is needed )
  61. Front
    Which antiretroviral drug class blocks HIV co-receptor (adhesion) interactions (e.g. maraviroc)?
    Back
    Co-receptor antagonists
  62. Front
    Which antiretroviral drug class blocks gp41-mediated membrane fusion (e.g. enfuvirtide)?
    Back
    Fusion inhibitors
  63. Front
    How do NRTIs (nucleoside/nucleotide reverse transcriptase inhibitors) work?
    Back
    They are incorporated into the growing DNA chain and terminate elongation (chain terminators)
  64. Front
    What was the prototype NRTI and first FDA-approved antiretroviral (1987)?
    Back
    Zidovudine (AZT)
  65. Front
    How do NNRTIs (e.g. efavirenz) inhibit reverse transcriptase ?
    Back
    They bind allosterically to reverse transcriptase and inactivate it
  66. Front
    Which antiretroviral drug class blocks integration of viral DNA (e.g. raltegravir, dolutegravir)?
    Back
    Integrase strand transfer inhibitors (InSTIs)
  67. Front
    Which antiretroviral drug class blocks post-translational polyprotein processing (e.g. darunavir)?
    Back
    Protease inhibitors
  68. Front
    What gives AZT its therapeutic selectivity?
    Back
    AZT-triphosphate has ~100-fold higher affinity for viral reverse transcriptase than for host DNA polymerases
  69. Front
    What serious side effects result from AZT 's residual inhibition of host polymerases?
    Back
    Anemia, neutropenia, neuropathy, and myopathy
  70. Front
    Why is AZT monotherapy avoided in favor of cART ?
    Back
    Monotherapy rapidly selects for resistant viral mutants
  71. Front
    What is the minimum drug requirement defining combination antiretroviral therapy ( cART )?
    Back
    At least two drugs from two different drug classes
  72. Front
    Why can cART manage but not eliminate HIV ?
    Back
    Dormant HIV reservoirs in long-lived cells are inaccessible to all current drugs
  73. Front
    What was the window period of a 1st-generation HIV ELISA , and which marker did it detect?
    Back
    ~8–10 weeks ; detected IgG only (using crude HIV antigen from infected-cell lysates)
  74. Front
    What did the 1st-generation HIV ELISA use as its capture antigen?
    Back
    Crude HIV antigen from infected-cell lysates
  75. Front
    How did the 2nd-generation HIV ELISA improve on the 1st-generation test?
    Back
    It used recombinant HIV proteins instead of crude lysate antigen, improving specificity
  76. Front
    Cloze: Across ELISA generations the window period shrinks: 1st-gen ~{{8–10 weeks}}, 3rd-gen ~{{3 weeks}}, 4th-gen ~{{2 weeks}}.
    Back
    1st-gen ~8–10 weeks; 3rd-gen ~3 weeks; 4th-gen ~2 weeks
  77. Front
    Why can a Western blot NOT reliably distinguish HIV-1 from HIV-2 ?
    Back
    HIV-1 and HIV-2 are distinct viruses with different protein (band) profiles , so their patterns overlap/confuse on the blot
  78. Front
    Clinically, why does confusing HIV-1 with HIV-2 matter (a key reason modern algorithms moved past Western blot)?
    Back
    The drug regimens differ between HIV-1 and HIV-2, so misidentification leads to errors in treatment selection
  79. Front
    How many distinct stages make up the HIV lifecycle?
    Back
    Eight
  80. Front
    List the eight stages of the HIV lifecycle in order.
    Back
    Binding → fusion → reverse transcription → nuclear transport → integration → replication → assembly/budding → maturation
  81. Front
    What term describes the HIV DNA once integrase has inserted it permanently into the host chromosome?
    Back
    Provirus
  82. Front
    After HIV infection, between which days does plasma HIV RNA reach its peak level?
    Back
    Days 20–30
  83. Front
    How is an HIV Western blot showing NO bands (beyond the negative control) interpreted?
    Back
    Negative
  84. Front
    In an HIV ELISA , after the patient's anti-HIV antibodies bind the coated HIV proteins, what is added next to enable detection?
    Back
    An enzyme-linked secondary antibody (anti-human IgG/IgM)
  85. Front
    What generates the measurable signal at the final step of an HIV ELISA ?
    Back
    A substrate added to the enzyme-linked secondary antibody , producing a colorimetric/luminescent signal proportional to bound target
  86. Front
    Name the four phases of the RT-qPCR amplification plot in order.
    Back
    Baseline → exponential → linear → plateau
  87. Front
    In the RT-qPCR amplification plot, during which phase is fluorescence too low to distinguish from background?
    Back
    The baseline phase
  88. Front
    What happens during the linear phase of an RT-qPCR amplification plot?
    Back
    Amplification continues but slows as reagents begin to deplete (efficiency drops below doubling)
  89. Front
    What is the starting material for the laboratory RT-PCR gene-expression workflow?
    Back
    Total RNA extracted from cells
  90. Front
    Besides HIV viral load, name two clinical/laboratory applications of RT-PCR .
    Back
    Diagnosis of RNA viruses (e.g., COVID-19, influenza) , detection of cancer mutations, treatment-response monitoring, gene-expression analysis, or pharmaceutical QC (any two)
  91. Front
    Why does a PCR program begin with a longer initial denaturation step (4 min at 95°C)?
    Back
    To fully separate the template and activate the polymerase before cycling begins
  92. Front
    Why does a PCR program end with a final extension step (7 min at 72°C)?
    Back
    To complete any partially synthesized strands
  93. Front
    Which antibody class provides the body's first-line, short-term response to a new antigen?
    Back
    IgM
  94. Front
    What is the active intracellular form of AZT (zidovudine) that actually inhibits reverse transcriptase ?
    Back
    AZT-triphosphate (AZT must be phosphorylated to its triphosphate to act as a chain terminator)
  95. Front
    The gp120-CD4 binding interaction determines what property of HIV (which cells it can infect)?
    Back
    Viral tropism

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