[Microbiology] Atlas about Applications of Molecular Diagnostics

Applications of Molecular Diagnostics, Atlas about Applications of Molecular Diagnostics, atlas in microbiology, atlas in medical, tuyenlab.net

Three different oligonucleotide hybrids.
Fig 1.  Three different oligonucleotide hybrids. A, DNA:DNA hybrid. B, DNA:RNA hybrid. The top strand is DNA and the bottom strand is RNA; note the “U”s instead of “T”s. C, RNA:RNA hybrid.


Southern blot
Fig 2.  Southern blot. A, Chromosomal DNA fragments separated in an agarose gel are transferred to a solid membrane. B, Labeled probe specific for a nucleic acid target is incubated with the separated DNA on the membrane. C, Excess probe is washed from the membrane, leaving probe bound only to the appropriate target DNA. D, The probe:target DNA hybrid is detected.

One polymerase chain reaction cycle.
Fig 3.  One polymerase chain reaction cycle. A, Template DNA is denatured by heat to yield two single-stranded DNA strands. B, The temperature of the reaction is cooled and the two single-stranded primers (Primer 1 and 2, in blue) anneal to the template DNA strands. DNA polymerase (blue spheres, labeled Pol). C, The temperature of the assay is heated to 72° C, and DNA polymerase adds nucleotides to the primers to synthesize new double-stranded DNA molecules (new DNA in red) in the primer extension step. The new DNA is then used for the next cycle.

Work flow diagram for a polymerase chain reaction (PCR) laboratory.
Fig 4.  Work flow diagram for a polymerase chain reaction (PCR) laboratory. Room A: Reagent preparation room for PCR components; this is a clean room with a laminar flow hood where no template nucleic acid is allowed. Room B: Nucleic acid extraction room with a biologic safety cabinet for clinical specimens and contamination control. Room C: Thermal cycling room where specimens are amplified. Room D: Agarose gel electrophoresis detection room, if necessary. BSC, Biologic safety cabinet; FH, flow hood.

Agarose gel electrophoresis
Fig 5.  Agarose gel electrophoresis. An agarose gel is shown submerged in running buffer in a gel box; next to the gel box is a voltage source that supplies the electric current to separate nucleic acids. The blue loading dye is visible in individual samples after migrating partway through the agarose gel matrix. Wells are visible at the top of the gel (at left in this image) for loading samples.

Fig 6.  Ethidium bromide–stained polymerase chain reaction (PCR) amplicons separated in an agarose gel. The image was obtained with an ultraviolet (UV) lamp. PCR was used to amplify a gene from unknown human papillomavirus (HPV) from clinical samples. Lane M is a 100-bp ladder; the 100-bp and 400-bp bands are indicated by arrows to the left of the image. Wells in which the samples were loaded are indicated by the arrow on the top right of the image, and the loading buffer dye front is indicated at the bottom right. Lane 1: Negative control (dH2O instead of template DNA). Lane 2: HPV-positive control. Lanes 3 to 6: Unknown samples. The unknown sample in lane 6 was positive for HPV. Nonspecific amplicons can be observed in lanes 3 and 5.

 Ethidium bromide–stained polymerase chain reaction (PCR) amplicons separated in an agarose gel.
Fig 7.  Ethidium bromide–stained polymerase chain reaction (PCR) amplicons separated in an agarose gel. The image was obtained with an imaging system. PCR was used
to amplify a gene from unknown human papillomavirus (HPV) from clinical samples. Lane M is a 100-bp ladder; the 100-bp and 400-bp bands are indicated by arrows to the left
of the image. Lanes 1 to 6 were assayed for HPV; lanes 8 to 13 were assayed for β-actin, an internal control that should be present in all human specimens. Lanes 1 and 8: Negative
control (dH2O instead of template DNA). Lanes 2 and 9: HPV-positive control (also a positive control for β-actin). Lanes 3 to 6 and 10 to 13: Unknown samples. Lane 7: Empty lane. The unknown sample in lane 4 was positive for HPV. β-Actin was present in all unknown samples.

Fig 8.  Real-time PCR data analysis for herpes simplex virus (HSV) from a human
specimen. Fluorescent peaks are depicted as fluorescence intensity (Y-axis) versus PCR
cycle number (X-axis). A positive control (HSV PC) was assayed along with an internal
control, β-actin, which should be present in all human samples. The HSV PC and the
β-actin positive control (β-actin PC) have fluorescent peaks, as does the β-actin assay from
the unknown specimen. The unknown specimen did not have a peak for HSV. In
addition, a sample no template control (SNTC) was assayed for both HSV and β-actin,
and there are no fluorescent peaks for either.

The LightCycler (Roche Diagnostics), a real-time polymerase chain reaction system.
Fig 9.  The LightCycler (Roche Diagnostics), a
real-time polymerase chain reaction system.

The SmartCycler (Cepheid Instruments), another real-time polymerase chain reaction platform.
Fig 10. The SmartCycler (Cepheid Instruments),
another real-time polymerase chain reaction platform.

Fig 11.  The 5′ nuclease assay (Taqman). A, Primer and probe are annealed to template DNA. DNA polymerase (P) starts to extend from the primer. B, DNA polymerase cleaves the fluorescent dye (F) from the probe and from the quencher. Fluorescence is observed. C, As DNA polymerase continues to extend and synthesize a new strand of DNA, the probe is fragmented, and the fluorescent dye and the quencher are fully released from each other. Fluorescence accumulates as fluorescent dye molecules are released.

Fig 12.  Dual-probe fluorescence resonance energy transfer. A, Two labeled probes anneal to polymerase chain reaction (PCR) product as it accumulates. One probe is
labeled with a donor fluorescent dye (D) on the 3′ end, while the other probe is labeled on the 5′ end with an acceptor dye (A). The two probes anneal to the PCR product
head-to-tail. A single strand of PCR product is shown in this diagram after denaturation has occurred. B, The light source from the real time-PCR platform excites the donor
fluorescent dye. The donor then transfers this energy to the acceptor dye. The acceptor dye is excited and emits fluorescent light that is read by the instrument. Fluorescence
increases as PCR product accumulates
.

Fig 13.   Molecular beacons. The molecular beacon probe is a complementary hairpin loop structure. A fluorescent dye is bound to the 5′ end of the hairpin and a
quencher is attached to the 3′ end. A loop structure at the top of the molecule is complementary to formed polymerase chain reaction (PCR) product. When the denaturation step of
PCR occurs, PCR product and molecular beacon probes dissociate; a single strand of a PCR amplicon is shown here. The beacon anneals to formed PCR product, then the
fluorescent dye is removed from the quencher molecule. Fluorescence increases as PCR product accumulates.

Fig 14.  Scorpion primer mechanism. A, A Scorpion primer is a probe and a primer in one molecule. It is a hairpin molecule labeled on the 5′ end with a fluorophore and on the 3′ end with a quencher. A short priming sequence is attached to the 3′ end also. The priming
sequence anneals to the target DNA. B, DNA polymerase synthesizes a new strand of DNA from the short priming sequence. C, Denaturation occurs, and the newly formed
DNA and the Scorpion primer dissociate. An internal portion of the Scorpion primer is complementary to the product just formed. This portion anneals to the PCR product and
separates the fluorophore from the quencher; fluorescence then accumulates.

Fig 15.  Multiplex polymerase chain reaction (PCR) products separated by agarose gel electrophoresis. Multiplex PCR for the ermA (139-bp) and ermC (190-bp) genes from
unknown Staphylococcus aureus isolates. Lane M is the 100-bp ladder; the 100-bp band is indicated by an arrow to the left of the image. Lane 1: Negative control (dH2O as the template). Lane 2: ermA-positive control (cloned PCR product). Lane 3: ermC-positive control (cloned PCR product). Lanes 4 to 13: Unknown MRSA isolates. Unknown isolates from lanes 6, 7, 9, 11, and 13 were positive for ermA, whereas the isolate from lane 12 was positive for ermC.

Nucleic acid sequence based amplification (NASBA)
Fig 16.  Nucleic acid sequence based amplification (NASBA). A, Primer 1 anneals
to target RNA. B, Reverse transcriptase synthesizes a DNA copy of the RNA template.
C, RNase H degrades the original RNA template. D, Primer 2 anneals to the DNA copy.
E, Reverse transcriptase synthesizes another DNA strand, resulting in double-stranded
DNA (dsDNA). F, T7 RNA polymerase synthesizes many copies of transcript using the
dsDNA as template. G, Primer 2 anneals to the synthesized transcripts. H, Reverse
transcriptase makes a DNA copy of the transcripts. I, RNase H degrades the transcript
copies and primer 1 anneals to the DNA copies. J, Reverse transcriptase synthesizes new
DNA strands, again resulting in dsDNA. K, The many copies of dsDNA are then used as
template by T7 RNA polymerase, which synthesizes even more transcript. This process
continues in a loop.

Fig 17.  Branched DNA detection. A, Capture probes attached to a surface anneal
to target nucleic acid. B, Target probes anneal to nucleic acid and to preamplifier probes.
C, Amplifier probes anneal to preamplifier probes, forming a branched DNA (bDNA)
structure. D, Label probes (with bound alkaline phosphatase [AP]) anneal to the bDNA
structure. A large, amplified signal is detected enzymatically when the AP substrate is
added.

Hybrid capture
Fig 18.  Hybrid capture. A, RNA probes are annealed to target DNA, resulting in RNA:DNA hybrids. B, The hybrids are bound to capture antibodies attached to a solid support
mechanism. C, Several alkaline-phosphatase (AP)–conjugated antibodies bind to hybrids. The substrate is added for the AP, and resulting light emission is captured by a
luminometer.

Fig 19.  Cycling probe technology. A, The DNA:RNA:DNA chimeric probe with a 5′ fluorescent dye (F, in green) and a 3′ quenching molecule (Q, in black) is incubated with the target nucleic acid; DNA is in red whereas RNA in the probe is in blue. B, The probe anneals to the target nucleic acid and forms a hybrid. C, RNase H digests the RNA in the chimeric probe. D, Digestion of the RNA releases the fluorescent dye from the vicinity of the quencher, resulting in fluorescence. A new probe molecule will then anneal to the same target nucleic acid molecule, and the process continues. Signal amplification results.

Fig 20.  Example of pulsed field gel electrophoresis (PFGE) analysis of Staphylococcus aureus strains. Lanes 1 and 5 are known control strains of a S. aureus isolate. Lanes 2 to 4 are unknown strains from different patients with S. aureus isolates. Lanes 2 and 3 have the same banding pattern after PFGE and are the same strains. The lane 4 isolate is an unrelated strain.

Fig 21.  Example of a dendrogram showing the genetic relationships among different Enterobacteriaceae based on 16S rRNA gene sequencing.




This is a part of the book : Textbook of Diagnostic Microbiology 4th edition 2011 of authors: Connie R. Mahon, Donald C. Lehman and George Manuselis. If you want to view the full content of the book and support author. Please buy it here: http://amzn.to/2ctxo02

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Atlas for Medical: [Microbiology] Atlas about Applications of Molecular Diagnostics
[Microbiology] Atlas about Applications of Molecular Diagnostics
Applications of Molecular Diagnostics, Atlas about Applications of Molecular Diagnostics, atlas in microbiology, atlas in medical, tuyenlab.net
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