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This chart has three columns labeled components, interactions and mictotiter results. In row A the components are the red blood cells which do not interact with anything and show no reaction in a microtiter result. The lack of reaction is seen as a small red dot in the center of the well. In row B the components are viruses and red blood cells. The viruses and red blood cells clump together and this is seen in a microtiter result as redness throughout the well. This is called hemagglutination. In row C, the components are viruses, red blood cells and antibodies. The viruses and antibodies clump together but the red blood cells do not clump with anything. This is again seen as no reaction; this is called hemagglutination inhibition.
This chart shows the possible outcomes of a hemagglutination test. Row A: Erythrocytes do not bind together and will sink to the bottom of the well plate; this becomes visible as a red dot in the center of the well. Row B: Many viruses have hemagglutinins that causes agglutination of erythrocytes; the resulting hemagglutination forms a lattice structure that results in red color throughout the well. Row C: Virus-specific antibody, the viruses, and the erythrocytes are added to the well plate. The virus-specific antibodies inhibit agglutination, as can be seen as a red dot in the bottom of the well. (credit: modification of work by Centers for Disease Control and Prevention)
  • What is the outcome of a positive HIA test?

Nucleic acid amplification test

Nucleic acid amplification tests (NAAT) are used in molecular biology to detect unique nucleic acid sequences of viruses in patient samples. Polymerase chain reaction (PCR) is an NAAT used to detect the presence of viral DNA in a patient’s tissue or body fluid sample. PCR is a technique that amplifies (i.e., synthesizes many copies) of a viral DNA segment of interest. Using PCR, short nucleotide sequences called primers bind to specific sequences of viral DNA, enabling identification of the virus.

Reverse transcriptase-PCR (RT-PCR) is an NAAT used to detect the presence of RNA viruses. RT-PCR differs from PCR in that the enzyme reverse transcriptase (RT) is used to make a cDNA from the small amount of viral RNA in the specimen. The cDNA can then be amplified by PCR. Both PCR and RT-PCR are used to detect and confirm the presence of the viral nucleic acid in patient specimens.

Hpv scare

Michelle, a 21-year-old nursing student, came to the university clinic worried that she might have been exposed to a sexually transmitted disease (STD). Her sexual partner had recently developed several bumps on the base of his penis. He had put off going to the doctor, but Michelle suspects they are genital warts caused by HPV. She is especially concerned because she knows that HPV not only causes warts but is a prominent cause of cervical cancer. She and her partner always use condoms for contraception, but she is not confident that this precaution will protect her from HPV.

Michelle’s physician finds no physical signs of genital warts or any other STDs, but recommends that Michelle get a Pap smear along with an HPV test. The Pap smear will screen for abnormal cervical cells and the CPEs associated with HPV; the HPV test will test for the presence of the virus. If both tests are negative, Michelle can be more assured that she most likely has not become infected with HPV. However, her doctor suggests it might be wise for Michelle to get vaccinated against HPV to protect herself from possible future exposure.

  • Why does Michelle’s physician order two different tests instead of relying on one or the other?

Enzyme immunoassay

Enzyme immunoassays (EIAs) rely on the ability of antibodies to detect and attach to specific biomolecules called antigens. The detecting antibody attaches to the target antigen with a high degree of specificity in what might be a complex mixture of biomolecules. Also included in this type of assay is a colorless enzyme attached to the detecting antibody. The enzyme acts as a tag on the detecting antibody and can interact with a colorless substrate, leading to the production of a colored end product. EIAs often rely on layers of antibodies to capture and react with antigens, all of which are attached to a membrane filter (see [link] ). EIAs for viral antigens are often used as preliminary screening tests. If the results are positive, further confirmation will require tests with even greater sensitivity, such as a western blot or an NAAT . EIAs are discussed in more detail in EIAs and ELISAs .

The explanation of EIA is separated to show what occurs in a positive sample and what occurs in a negative sample. First patient sample is applied to a membrane filter. If the sample contains viruses they are trapped by the filter. Next, antibody with enzyme conjugate is added. Antibody will attach to antigen if present. Next is a wash step. If the virus is present the enzyme binds to the virus, otherwise the enzyme washes away. Finally substrate is added. If the antibody is present (because it is bound to the virus) the attached enzyme causes a color change. If no enzyme linked antibody is present, no color change occurs.
Similar to rapid, over-the-counter pregnancy tests, EIAs for viral antigens require a few drops of diluted patient serum or plasma applied to a membrane filter. The membrane filter has been previously modified and embedded with antibody to viral antigen and internal controls. Antibody conjugate is added to the filter, with the targeted antibody attached to the antigen (in the case of a positive test). Excess conjugate is washed off the filter. Substrate is added to activate the enzyme-mediated reaction to reveal the color change of a positive test. (credit: modification of work by “Cavitri”/Wikimedia Commons)
  • What typically indicates a positive EIA test?

Part 3

Along with the RT/PCR analysis, David’s saliva was also collected for viral cultivation. In general, no single diagnostic test is sufficient for antemortem diagnosis, since the results will depend on the sensitivity of the assay, the quantity of virions present at the time of testing, and the timing of the assay, since release of virions in the saliva can vary. As it turns out, the result was negative for viral cultivation from the saliva. This is not surprising to David’s doctor, because one negative result is not an absolute indication of the absence of infection. It may be that the number of virions in the saliva is low at the time of sampling. It is not unusual to repeat the test at intervals to enhance the chance of detecting higher virus loads.

  • Should David’s doctor modify his course of treatment based on these test results?

Jump to the next Clinical Focus box. Go back to the previous Clinical Focus box.

Key concepts and summary

  • Viral cultivation requires the presence of some form of host cell (whole organism, embryo, or cell culture).
  • Viruses can be isolated from samples by filtration.
  • Viral filtrate is a rich source of released virions.
  • Bacteriophages are detected by presence of clear plaques on bacterial lawn.
  • Animal and plant viruses are detected by cytopathic effects , molecular techniques (PCR, RT-PCR), enzyme immunoassays, and serological assays (hemagglutination assay, hemagglutination inhibition assay).

Fill in the blank

Viruses can be diagnosed and observed using a(n) _____________ microscope.

Electron

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Cell abnormalities resulting from a viral infection are called ____________ _____________.

cytopathic effects

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Short answer

Briefly explain the various methods of culturing viruses.

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Questions & Answers

enlargement of the thyroid gland resulting in over production of hormone.
Kamal Reply
What can u say on Thyroid Cancer?
Abdulkareem
explain the Grave's disease
John Reply
what is cell
Avi Reply
is unit of life
Kamaluddeen
Ok
mohamed
who is an industrial microbiologist
Cynthia Reply
I want to know the biochemical composition of bacteria
Josh Reply
It contains peptidoglcon, DNA nd RNA
Asiya
bacteriophage disadvantage
Momina Reply
disease due to __________ abnormalities are termed primary immunodeficiencies
Tayee Reply
Some primary immunodeficiencies are due to a defect of a single cellular or humoral component of the immune system.
Prince
Examples of primary immunodeficiencies include: chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency etc
Prince
thank you
Nana
explain microbial mutation
Emerald
what is mutation
Cynthia Reply
alteration in genetic makeup
UDEME
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BELLO Reply
welcome on board
Kamaluddeen
Thanks brother I'm an undergraduate. I hope to study for MBBS.
BELLO
pls guys help me out
Linda
what is limitation of plate
Linda
limitation of plate load test . there are some that should be considered while performing load test which are given below, this test is usually performed on relatively similar plate ,usually 1 or 2 square foot area the reason is that the plate of greater are the economically not feasible
yemi
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Shafiu
thanks shafiu
gambo
how to differentiate Gram positive from Gram negative bacteria?
Faheem
by the cell membrane
Tim
with gram staining which has 4 steps
Freedom
Mechanism of bacterial pathogenicity
rashida Reply
Toxicity
Ibrahim
write a short note on Algea
najaatu Reply
green minutes plants organisms that are produced in turf.
Prince
Algae is a kind of a photosynthetic organism, which is usually grown in the moist areas. These are usually the simple plants that grow near to the water bodies. It contains a kind of chlorophyll pigments that act as a primary coloring agent.
Avi
they are eukaryotic and most lived in fresh water. they are photosynthetic that's, they contain chlorophyll and store starch in discrete chloroplast.
Prince
are bacteria important to man
just Reply
yes, depending on the type of bacteria .eg the normal florals, the latic acid batteries etc are important to man
Lois
I mean latic acid bacterial
Lois
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Jane Reply
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Rose
sahi kon hai bhosri ke
saurabh Reply
define metabolism of carbohydrates with example
Thavasi Reply
what is sterilization a
Sani
the process of keep equipment free from bacteria
shar
is it only bacteria?
Lois
no undesirable fungi and contamination also.
Khushbu
what is streak plate method
Offikwu
what is the biofilm
Dimingu
metabolism is the sum of all the biochemical reaction required for energy generation and use of that energy to synthesize cell materials from small molecules in environment.
Alex
boclenia
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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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