Molecular Detection of Microorganisms
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Questions and Answers

Which molecular technique is specifically used to confirm Hepatitis C and HIV infections?

Western blot technique.

What is the primary purpose of serological tests like ELISA in the context of viral infections?

To screen for the presence of antibodies against viruses.

Name two nucleic acid amplification methods used for detecting microorganisms.

PCR and loop-mediated isothermal amplification (LAMP).

What is the significance of using agarose gel electrophoresis in molecular detection?

<p>To separate and analyze nucleic acids based on size.</p> Signup and view all the answers

Which organisms are noted to be received in clinical laboratories in high volumes?

<p>Streptococcus, Neisseria, and Chlamydia.</p> Signup and view all the answers

What is the role of quantitative PCR (qPCR) in infectious disease detection?

<p>To quantify the amount of specific DNA or RNA in a sample.</p> Signup and view all the answers

Identify one gene associated with antimicrobial resistance mentioned in the content.

<p>mecA.</p> Signup and view all the answers

What challenge is mentioned regarding the processing of multiple microbial samples in clinical laboratories?

<p>Samples must be tested in batches due to high volumes.</p> Signup and view all the answers

What are the primary advantages of molecular-based tests for microorganism detection?

<p>Molecular-based tests provide higher sensitivity and specificity, enabling rapid and accurate detection of microorganisms.</p> Signup and view all the answers

Why are Histoplasma and Coccidioides considered hazardous to handle in clinical laboratories?

<p>Both Histoplasma and Coccidioides pose a high risk of infection and require high-level biosecurity measures due to their pathogenicity.</p> Signup and view all the answers

What role does the selection of target sequences play in nucleic acid tests?

<p>The selection of target sequences is crucial as it determines the specificity and effectiveness of the nucleic acid tests in detecting the intended microorganisms.</p> Signup and view all the answers

What challenges arise when culturing fungal organisms like Histoplasma and Coccidioides?

<p>Culturing these fungi is challenging due to their fastidious nature and the requirement for specialized laboratory conditions.</p> Signup and view all the answers

In the context of molecular detection, why is mycobacterium considered a difficult organism to grow?

<p>Mycobacterium is considered difficult to grow due to its fastidious nature and the lengthy incubation period required for growth.</p> Signup and view all the answers

How do molecular techniques improve the detection of microorganisms that are bioterrorism agents?

<p>Molecular techniques improve detection by allowing rapid identification and analysis of these microorganisms with higher sensitivity.</p> Signup and view all the answers

What types of organisms are highlighted as included in the molecular detection framework?

<p>The framework includes bacteria, viruses, parasites, and fungi specifically focusing on those challenging to culture.</p> Signup and view all the answers

What are chromosomal abnormalities, and why are they significant in molecular detection of inherited diseases?

<p>Chromosomal abnormalities refer to structural changes in chromosomes that can lead to genetic disorders, making them significant for diagnosis in inherited diseases.</p> Signup and view all the answers

What is the primary purpose of multiplex PCR methods?

<p>To simultaneously detect multiple parasites in a sample.</p> Signup and view all the answers

Name two parasites that can be detected simultaneously using multiplex real-time PCR assays.

<p>Entamoeba histolytica and Giardia lamblia.</p> Signup and view all the answers

What additional feature do multiplex PCR methods include to ensure accuracy?

<p>An internal control to determine PCR efficiency and detect inhibition.</p> Signup and view all the answers

How do bacteria produce resistance to antimicrobial agents according to the content?

<p>By producing enzymes that inactivate the agents or altering target binding sites.</p> Signup and view all the answers

What is the role of β-lactamase in bacterial resistance?

<p>It cleaves the β-lactam ring of β-lactam antimicrobials, rendering them inactive.</p> Signup and view all the answers

Describe a mechanism through which microorganisms can alter drug accessibility.

<p>Through active extrusion of the drug from the microbial cell.</p> Signup and view all the answers

What kind of samples are mentioned as likely to have PCR inhibition?

<p>Stool samples.</p> Signup and view all the answers

What is a common example of an organism that develops resistance through the production of β-lactamase?

<p>Staphylococcus aureus.</p> Signup and view all the answers

What is the clinical significance of a positive PCR assay for respiratory tract pathogens?

<p>The clinical significance is questionable due to high colonization rates in the population, especially in children, which makes it difficult to distinguish between colonization and active infection.</p> Signup and view all the answers

What are the common non-molecular methods used for bacterial identification in respiratory specimens?

<p>Common non-molecular methods include biochemical tests, which are often time-consuming and lack sensitivity.</p> Signup and view all the answers

What distinguishes an infection caused by a respiratory pathogen from mere colonization?

<p>An infection causes harm and presents signs and symptoms, while colonization occurs without causing any harm to the host.</p> Signup and view all the answers

What are the target genomes for qPCR detection of Bordetella pertussis?

<p>The targets for qPCR detection are IS481 and IS1001 regions of the Bordetella genome.</p> Signup and view all the answers

What causes aneuploidy during gamete production?

<p>Erroneous separation of chromosomes during egg or sperm production leads to aneuploidy.</p> Signup and view all the answers

What limitation does 16S RNA sequencing have in identifying Streptococcus species?

<p>16S RNA sequencing cannot differentiate among alpha hemolytic Streptococcus species due to their more than 99% sequence homology.</p> Signup and view all the answers

What are the consequences of autosomal trisomy?

<p>Autosomal trisomy results in three copies of a chromosome instead of the normal two.</p> Signup and view all the answers

Why is autosomal monosomy generally incompatible with life?

<p>Autosomal monosomy typically results in lethal developmental issues due to the lack of essential genetic material.</p> Signup and view all the answers

Why is molecular-based assays preferred over traditional methods in identifying respiratory tract pathogens?

<p>Molecular-based assays are preferred because they can directly detect and identify bacterial pathogens in specimens, providing quicker and more accurate results.</p> Signup and view all the answers

What type of specimens are commonly used for testing respiratory tract pathogens?

<p>Common specimens include bronchoalveolar lavage (BAL), bronchial washings, and throat swabs.</p> Signup and view all the answers

How does antibiotic exposure impact mutation and selection processes in bacteria?

<p>Antibiotic exposure can stimulate mutation and selection, allowing bacteria with advantageous mutations to survive and proliferate.</p> Signup and view all the answers

What role does the mecA gene play in methicillin-resistant S. aureus (MRSA)?

<p>The mecA gene encodes PBP2a, which has a low affinity for methicillin, contributing to MRSA's resistance.</p> Signup and view all the answers

How is Bordetella pertussis primarily transmitted?

<p>Bordetella pertussis is transmitted via direct contact with infected respiratory secretions.</p> Signup and view all the answers

What distinguishes mosaicism from chimerism?

<p>Mosaicism consists of genetically distinct cell populations from one zygote, while chimerism involves multiple zygotes.</p> Signup and view all the answers

What is the significance of karyotyping for chromosome mutations?

<p>Karyotyping allows the visualization of chromosome structures larger than 4 million base pairs to identify abnormalities.</p> Signup and view all the answers

Why is sex chromosome aneuploidy more frequently tolerated than autosomal aneuploidy?

<p>Sex chromosome aneuploidy is often tolerated because it usually has less severe effects on development and viability.</p> Signup and view all the answers

What is the result of having a heterozygous specimen for the + /m genotype?

<p>It yields a combination of the normal and mutant pattern.</p> Signup and view all the answers

What is the primary consequence of mutations in the MTHFR gene?

<p>It causes hyperhomocysteinemia due to a deficiency in 5,10-methylenetetrahydrofolate reductase.</p> Signup and view all the answers

Which genes are associated with hemochromatosis and what is their role?

<p>The HFE or HLA-H gene is associated, causing dysfunction that leads to iron overabsorption.</p> Signup and view all the answers

What mutation is most commonly observed in patients with hemochromatosis?

<p>The most frequently observed mutation is C282Y/Cys282Tyr.</p> Signup and view all the answers

How can mutations related to hemochromatosis be identified?

<p>They can be detected using PCR-RFLP methods.</p> Signup and view all the answers

What are the health implications of untreated hemochromatosis?

<p>It can lead to pancreas, liver, and skin damage, heart disease, and diabetes.</p> Signup and view all the answers

What initial test is typically performed to detect lysosomal storage diseases?

<p>A biochemical screening test for loss of enzyme activity is usually conducted.</p> Signup and view all the answers

What is the underlying defect in cystic fibrosis?

<p>Cystic fibrosis is caused by mutations in the CFTR gene, affecting chloride ion transport.</p> Signup and view all the answers

Study Notes

Molecular Detection of Microorganisms

  • Molecular methods are used to detect and identify organisms that are difficult to isolate, hazardous to handle, or require high throughput.
  • Nucleic acid-based hybridization and amplification procedures are used to detect microorganisms.
  • Examples include PCR, TMA, and LAMP.
  • Real-time PCR (qPCR) is frequently used for infectious agents due to its sensitivity, safety, closed-tube format, and quantitative information.
  • Sequencing is used to identify new organism variants.
  • Immunoassays are used for rapid testing of microorganisms
  • Hybridization techniques such as Southern and Northern blots detect protein and nucleic acid particles, respectively.
  • Mass spectrometry determines amino acid sequences of proteins from microorganisms or cells within a disease.

Selection of Sequence Targets

  • Sequence targets for microorganisms are available from the NCBI or published literature.
  • Primer or probe specificity depends on target genome location.
  • Sequences are often shared between microorganisms, especially in closely related species.
  • Choosing specific sequences allows for the detection of particular strains/variants instead of general microbial identification.
  • Targeting conserved genes in microorganisms, such as HIV, is useful for identifying sequences for nucleic acid tests.

Detection of Bacteria

  • Bacteria causing respiratory tract infections, such as Bordetella, Legionella, Mycobacteria, Chlamydia, and Streptococcus species, are frequently tested for.
  • Non-molecular methods are often less sensitive.
  • Molecular-based assays allow direct detection and identification in respiratory specimens.
  • Individual diagnostic tests are available for various bacterial pathogens, such as Bordetella pertussis.
  • Legionella pneumophila and Mycobacterium tuberculosis detection utilize specific PCR assays targeting unique regions of their genomes.
  • Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Streptococcus pneumoniae testing often uses PCR, sequencing or MALDI technologies.

Detection of Viruses

  • Using molecular methods helps diagnose or monitor viral infections.
  • Target amplification assays such as PCR, RT-PCR, qPCR, and TMA, as well as signal amplification assays like bDNA amplification and hybrid capture are used for viral infections.
  • Viruses that exhibit rapid mutation are better diagnosed/monitored with nucleic acid amplification assays.
  • Nucleic Acid Amplification assays are essential for clinical virology labs.
  • Identifying specific sequences for viruses using RT-PCR facilitates detection/characterization.

Human Immunodeficiency Virus (HIV)

  • HIV infection is diagnosed using antibodies, western blots, and nucleic acid amplification assays (PCR, NASBA, bDNA).
  • Viral load is measured to evaluate treatment effectiveness and guide therapeutic management.

Herpes Viruses

  • Herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella-zoster virus (VZV) are targeted for diagnosis using PCR tests.
  • Viral culture, antigen tests, and antibody tests are used in some cases, especially if reactivation is suspected.

Hepatitis C Virus (HCV)

  • Diagnosis and monitoring of HCV involves antibody testing followed by nucleic acid amplification assays like RT-PCR, TMA, and bDNA.
  • HCV genotyping is carried out using PCR-RFLP, reverse hybridization, DNA sequencing, or PCR-MCA.

Human Papilloma Virus (HPV)

  • HPV is diagnosed using hybridization and amplification methods.
  • Certain HPV types are associated with cancer, and vaccination plays an important role in prevention.

Respiratory Syncytial Virus (RSV)

  • Molecular assays for RSV A and B, influenza A and B, and parainfluenza viruses are used for the detection and characterization of respiratory tract infections.

BK and JC Viruses

  • BK and JC viruses, which are human polyomaviruses, are detected with PCR tests.

Mycology

  • Molecular-based methods allow for more sensitive and specific detection and identification of fungi.
  • Broad-range PCR and subsequent analysis are used for clinical analysis.

Parasites

  • Molecular tests targeting specific parasites like Trypanosomes, Plasmodia, Toxoplasma, Entamoebae, and Cryptosporidium are useful for diagnosis.
  • These tests allow for quick and sensitive identification from various specimen sources.

Resistance to Antimicrobial Agents

  • Microorganisms develop resistance through enzyme production, target alterations, or changes in drug transport.
  • Examples include beta-lactamases and altered penicillin-binding proteins.

Inherited Diseases: Chromosome Abnormalities

  • Karyotyping, flow cytometry, and FISH (fluorescence in situ hybridization) are used.
  • Structural mutations, like deletions, duplications, inversions, and translocations that are >4 million base pairs (bp) in size are identifiable through karyotyping.

Inherited Diseases: Single-Gene Disorders

  • Penetrance and expressivity variability are characteristic of these disorders.
  • Genes for structural, receptor, regulatory, and enzymatic proteins are affected.

Inherited Disease: Mitochondrial Disorders

  • Mutations in mitochondrial DNA are maternally inherited.
  • Techniques like Southern blot and PCR-RFLP are employed.
  • Disorders include subacute necrotizing encephalomyelopathy, Leigh syndrome, etc.

Inherited Diseases: Nucleotide Repeat Expansion Disorders

  • Nucleotide repeats, including triplet repeats, can cause genetic diseases.
  • Fragile X syndrome and Huntington's disease are examples.
  • PCR is used to detect repeat expansions in affected individuals and carriers.

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Description

This quiz covers various molecular methods used for detecting and identifying microorganisms that are challenging to isolate. Topics include nucleic acid hybridization, PCR, real-time PCR, immunoassays, and mass spectrometry. Test your knowledge on the techniques and applications in the field of microbiology.

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