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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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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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