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The National Center for Biotechnology Information houses a widely used genetic sequence database called GenBank where researchers deposit genetic information for public use. Upon publication of sequence data, researchers upload it to GenBank, giving other researchers access to the information. The collaboration allows researchers to compare newly discovered or unknown sample sequence information with the vast array of sequence data that already exists.

Using a naat to diagnose a C. difficile Infection

Javier, an 80-year-old patient with a history of heart disease, recently returned home from the hospital after undergoing an angioplasty procedure to insert a stent into a cardiac artery. To minimize the possibility of infection, Javier was administered intravenous broad-spectrum antibiotics during and shortly after his procedure. He was released four days after the procedure, but a week later, he began to experience mild abdominal cramping and watery diarrhea several times a day. He lost his appetite, became severely dehydrated, and developed a fever. He also noticed blood in his stool. Javier’s wife called the physician, who instructed her to take him to the emergency room immediately.

The hospital staff ran several tests and found that Javier’s kidney creatinine levels were elevated compared with the levels in his blood, indicating that his kidneys were not functioning well. Javier’s symptoms suggested a possible infection with Clostridium difficile , a bacterium that is resistant to many antibiotics. The hospital collected and cultured a stool sample to look for the production of toxins A and B by C. difficile , but the results came back negative. However, the negative results were not enough to rule out a C. difficile infection because culturing of C. difficile and detection of its characteristic toxins can be difficult, particularly in some types of samples. To be safe, they proceeded with a diagnostic nucleic acid amplification test (NAAT). Currently NAATs are the clinical diagnostician’s gold standard for detecting the genetic material of a pathogen. In Javier’s case, qPCR was used to look for the gene encoding C. difficile toxin B ( tcdB ). When the qPCR analysis came back positive, the attending physician concluded that Javier was indeed suffering from a C. difficile infection and immediately prescribed the antibiotic vancomycin , to be administered intravenously. The antibiotic cleared the infection and Javier made a full recovery.

Because infections with C. difficile were becoming widespread in Javier’s community, his sample was further analyzed to see whether the specific strain of C. difficile could be identified. Javier’s stool sample was subjected to ribotyping and repetitive sequence-based PCR (rep-PCR) analysis. In ribotyping, a short sequence of DNA between the 16S rRNA and 23S rRNA genes is amplified and subjected to restriction digestion ( [link] ). This sequence varies between strains of C. difficile , so restriction enzymes will cut in different places. In rep-PCR, DNA primers designed to bind to short sequences commonly found repeated within the C. difficile genome were used for PCR. Following restriction digestion, agarose gel electrophoresis was performed in both types of analysis to examine the banding patterns that resulted from each procedure ( [link] ). Rep-PCR can be used to further subtype various ribotypes, increasing resolution for detecting differences between strains. The ribotype of the strain infecting Javier was found to be ribotype 27, a strain known for its increased virulence, resistance to antibiotics, and increased prevalence in the United States, Canada, Japan, and Europe. Patrizia Spigaglia, Fabrizio Barbanti, Anna Maria Dionisi, and Paola Mastrantonio. “ Clostridium difficile Isolates Resistant to Fluoroquinolones in Italy: Emergence of PCR Ribotype 018.” Journal of Clinical Microbiology 48 no. 8 (2010): 2892–2896.

  • How do banding patterns differ between strains of C. difficile ?
  • Why do you think laboratory tests were unable to detect toxin production directly?
A gel showing various bands of various PCR-ribotypes. The bottom lane is labeled Javier’s an matches the banding pattern from PCR-ribotype 027.
A gel showing PCR products of various Clostridium difficile strains. Javier’s sample is shown at the bottom; note that it matches ribotype 27 in the reference set. (credit: modification of work by American Society for Microbiology)
A diagram of molecular analysis. A circular bacterial genome is shown. Primers bind to repetitive sequences found at multiple locations in the genome. These regions are amplified using PCR. Gel electrophoresis is used to identify the size of the amplified regions. Amplified fragment patters can be compared to known samples to identify strains.
Strains of infectious bacteria, such as C. difficile, can be identified by molecular analysis. PCR ribotyping is commonly used to identify particular C. difficile strains. Rep-PCR is an alternate molecular technique that is also used to identify particular C. difficile strains. (credit b: modification of work by American Society for Microbiology)
  • How is PCR similar to the natural DNA replication process in cells? How is it different?
  • Compare RT-PCR and qPCR in terms of their respective purposes.
  • In chain-termination sequencing, how is the identity of each nucleotide in a sequence determined?

Key concepts and summary

  • Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon (typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample.
  • Agarose gel electrophoresis allows for the separation of DNA molecules based on size.
  • Restriction fragment length polymorphism (RFLP) analysis allows for the visualization by agarose gel electrophoresis of distinct variants of a DNA sequence caused by differences in restriction sites.
  • Southern blot analysis allows researchers to find a particular DNA sequence within a sample whereas northern blot analysis allows researchers to detect a particular mRNA sequence expressed in a sample.
  • Microarray technology is a nucleic acid hybridization technique that allows for the examination of many thousands of genes at once to find differences in genes or gene expression patterns between two samples of genomic DNA or cDNA,
  • Polyacrylamide gel electrophoresis (PAGE) allows for the separation of proteins by size, especially if native protein charges are masked through pretreatment with SDS.
  • Polymerase chain reaction allows for the rapid amplification of a specific DNA sequence. Variations of PCR can be used to detect mRNA expression ( reverse transcriptase PCR ) or to quantify a particular sequence in the original sample (real-time PCR ).
  • Although the development of Sanger DNA sequencing was revolutionary, advances in next generation sequencing allow for the rapid and inexpensive sequencing of the genomes of many organisms, accelerating the volume of new sequence data.

Fill in the blank

The __________ blot technique is used to find an RNA fragment within a sample that is complementary to a DNA probe.

northern

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The PCR step during which the double-stranded template molecule becomes single-stranded is called _____________.

denaturation

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The sequencing method involving the incorporation of ddNTPs is called __________.

Sanger sequencing, dideoxy method, or chain termination method

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True/false

In agarose gel electrophoresis, DNA will be attracted to the negative electrode.

false

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Short answer

Why is it important that a DNA probe be labeled with a molecular beacon?

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When separating proteins strictly by size, why is exposure to SDS first required?

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Why must the DNA polymerase used during PCR be heat-stable?

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Questions & Answers

write the life cycle of HIV
Firomsa Reply
describe the internal and external structure of prokaryotic cell in terms of there appearance and functions
Lenia Reply
compare and contrast similar structures found in prokaryotic and eukaryotic cells
Lenia
control of microorganisms
ISTEFAZUL Reply
how can a doctor treat a person affected by endospore forming bacteria in his/her wound?
Mambo Reply
a 28 years old woman come to your clinic with complain of fever painful genital blisters which express clear fluid when ruptured burning sensation around the bristers what is the diagnosis?
Ramadhani Reply
genital herpes caused by a virus called herpe simplex virus
Doris
what is the reference between selective medium and differential medium?
Tony Reply
the micro flora of air is transient why
Princess
Hello.... Am new here
essien Reply
welcome essien
Muhammad
welcome essien
Beka
classification of gram positive
lissa Reply
classify gram positive
lissa
catalase test is done to differentiate between staph and strep. as staph is catalase positive while strep is catalase negative then staph is differentiate further on coagulase positive and coagulase negative. staph aureus is coagulase positive while staph epidermidis is coagulase negative.
Muhammad
gram positive staph is further differentiated on sensitivity test and manitol salt fermentation test while gram positive strep is differentiated on hemolysis pattern
Muhammad
more about gramm positive
lissa
please
lissa
ok
Blessing
am back any gist
Blessing
taxonomy
Rahul
can i use the graph of bacterial growth for my master's thesis?
Christoph Reply
facultative anaerobic bavteria gives uniform turdibity in nutrient broth why?
Princess Reply
oils and waxes are not sterilized in autoclave
Princess
what is the function of paraffin
Muhammad
@ MAHI because they can grow all over the media from surface to the bottom as they can utilize oxygen or conduct fermentation in its absence.
Online
2.@MAHI Autoclave uses steam under pressure. The steam cannot penetrate through oil and wax. Thus, dry heat sterilization is preferred than autoclave .
Online
thnku
Nadiya
how nutirent agar can converted into blood agar
Princess
thank you
Princess
take a nutrient agar ........and add 5ml blood and put in it ...........!
Nadiya
nutrient agar +blood 5ml+ distilled water =blood agar .
Nadiya
mixed wellllll
Nadiya
hi
Rahul
why agar is not a neutrient source?
Shimul
because it's a general purpose agar only use for growing bacteria
Richa
way is antigen
opaleye
what is antigen
opaleye
antigen is a foreign body that cause activation of antibody?
ASNAKE
Antigen is a toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies.
Shimul
thanks for that
opaleye
what is pharmacology
Luyenu
a science that studies about the drug
ASNAKE
hello ever one
Sayid
y
Abdul
Work hard
Anigor
y
Abdul
thanks alot
Luyenu
What is the major different between gram positive and gram negative bacteria.
Asikur
bacteria kya hai
Mala
content of their cell wall
Doris
koi mujhse basic microbiology ki study k Lea book suggest kro .
Richa
Harley prescott
Vikas
what are the five predisposing factors of opportunistic infections
Asher Reply
1. long term exposure to diseases, that the body immunity weakens.
prosper
2. Nature Of some diseases to weaken body immunity such as, AIDS.
prosper
3. Use of immuno-suppresive drugs.
prosper
anyone... need a hand here 🙋😞
prosper
Low immune is the major cause eg for kaposis sarcoma
Davismith
suitable conditions e.g temp,
Muhammad
what os gnormal flora
kifayat Reply
morphological and genetic classification of bacteria
Potter Reply
what is the morphogical
Bupe
h
Sudhir
morphologycal is defined as relating to the branch of biology that deals with the form of living organisms,and with relationship between their structures.
Nadiya
relating to the form or structure of things .
Nadiya
what is the meaning of lessions
oluwa
i am frim Afghanistan.
wahidullah Reply
iam from iraq
Haidar
and i'm from pakistan
Muhammad
Nice
Amina
what's about the qualification
Muhammad
Hlo
Asiya
Hmm
Number
Almustafa Muhammad from Nigeria
Mujtafa
am ustaz Abdulsalam from Ghana
ustaz
am Bupe Chifita from Zambia
Bupe
what is microbiology
Bupe
is the study of invisible micro organism
Mujtafa
microbiology is the study of very too small organisms which we can't see with naked eyes ......in other words ....study of microorganism is called microbiology.
Nadiya
Hello! How Can Microorganisms Be Isolated From The Skin?.. 5 ways if possible. (briefly)
prosper
from skin swabbing is the prefer method worldwide
Muhammad
thanks Muhammad Nauman very much!
prosper
hi
Meek
hi Meek
prosper
are there any other ways pals?
prosper
hi prosper
Meek
hello Meek Mild.
prosper
Any question or ideas, Meek?😊
prosper
and if a patient have any blister or abscess on the skin then first cut the blister or abscess. inside material is taking by syringe.
Muhammad
in case of fingernail or toenails simply cut a small piece of nail and culture on the appropriate media at a required temperature and time
Muhammad
Again, thanks Mr. Muhammad! I appreciate the help.
prosper
you welcom prosper 😊
Muhammad
what are the media use for skin swab?
Sieh-Mlanwin
blood and macckonkey mostly usededia for skin bacteria
Muhammad
used media*
Muhammad
@ SIEH mostly fungal infection occurs in skin so SDA ( Sabouraud Dextrose Agar) or MEA ( Malt extract agar) are common.
Online
thanks
Sieh-Mlanwin
Microbiologicam source of vitamins
Bachi Reply
what do you mean?
Md
that's example?
Md
hi everyone
Muhammad
such as. c/s media
Md
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Md
just tell me about that ?
Md
where are u from
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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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