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Cycle 2

Repeat steps 1 through 3

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25-45 cycles resulting in 106-10B copies of target sequence

Conventional PCR involves 25 to 50 repetitive cycles, with each cycle comprising three sequential reactions: denaturation of target nucleic acid, primer annealing to single-stranded target nucleic acid, and extension of primer-target duplex.

Extraction and Denaturation of Target Nucleic Acid.

For PCR, nucleic acid is first extracted (released) from the organism or a clinical sample that potentially contains the target organism by heat, chemical, or enzymatic methods. Numerous manual methods are available to accomplish this task including a variety of commercially available kits that will extract either RNA or DNA, depending on the specific target of interest. In addition, other commercially available kits are designed to extract nucleic acids from specific types of clinical specimens such as blood or tissues. Most recently, automated instruments have been introduced for the extraction of nucleic acid from various sources (e.g., bacteria, viruses, tissue, blood).

Once extracted, target nucleic acid is then added to the reaction mix that contains all the necessary components for PCR to occur (primers, covalent ions, buffer, and enzyme) and then placed into a thermal cycler to undergo amplification (Figure 8-7). For PCR to begin, target nucleic acid must first be in the single-stranded conformation for the second reaction, primer annealing, to occur. Denaturation to a single strand, which is not necessary for RNA targets, is accomplished by heating to 94° C (see Figure 8-7). Of note, for many PCR procedures, especially those involving commonly encountered bacterial pathogens, disruption of the organism to release DNA is done in one step by heating the sample to 94° C.

Primer Annealing. Primers are short, single-stranded sequences of nucleic acid (i.e., oligonucleotides usually 20 to 30 nucleotides long) that are selected to specifically hybridize (anneal) to a particular nucleic acid target, essentially functioning like probes. As noted for hybridization tests, the abundance of available gene sequence data allows for the design of primers specific for a number of microbial pathogens and their virulence or antibiotic resistance genes. Thus, primer nucleotide sequence design depends on the intended target, such as genus-specific genes, species-specific genes, virulence genes, or antibiotic-resistance genes.

Primers are designed to be used in pairs that flank the target sequence of interest (see Figure 8-7). When the primer pair is mixed with the denatured target DNA, one primer anneals to a specific site at one end of the target sequence of one target strand while the other primer anneals to a specific site at the opposite end of the other, complementary target strand. Usually, primers are designed so that the distance between them on the target DNA is 50 to 1000 base pairs. The annealing process is conducted at 50° to 58° C, or higher. Once the duplexes are formed the last step in the cycle, which mimics the DNA replication process, begins.

Extension of Primer-Target Duplex. Annealing of primers to target sequences provides the necessary template format that allows DNA polymerase to add nucleotides to the 3' terminus (end) of each primer and produce by extension a sequence complementary to the target template (see Figure 8-7). Taq polymerase is the enzyme commonly used for primer extension, which occurs at 72° C. This enzyme is used because of its ability to function efficientiy at elevated temperatures and withstand die denaturing temperature of 94° C through several cydes. The ability to allow primer annealing and extension to occur at elevated temperatures without detriment to the polymerase increases the stringency of the reaction, thus decreasing the chance for amplification of nontarget nudeic acid (i.e., nonspecific amplification).

All three reaction components of PCR occur in the same tube that contains a mixture of target nudeic add, primers, components to optimize polymerase activity (i.e., buffer, cation [MgCl2], salt), and deoxy-nudeotides. To maintain continuous reaction cydes programmable thermal cyclers are used. These cyders hold the reaction vessel and carry the PCR mixture through each reaction step at the predse temperature and for the optimal duration.

As shown in Figure 8-7, for each target sequence originally present in the PCR mixture, two double-stranded fragments containing the target sequence are produced after one cycle. At the beginning of the second cyde of PCR, denaturation then produces four templates to which the primers will anneal. Following extension at the end of the second cyde, there will be four double-

stranded fragments containing target nudeic add. Therefore, with completion of each cyde there is a doubling of target nudeic add and after 30 to 40 cydes 107 to 108 target copies will be present in the reaction mixture.

Although it is possible to detect one copy of a pathogen's gene in a sample or patient specimen by PCR technology, detection is dependent on ¿be ability of the primers to locate and anneal to the single target copy and on optimum PCR conditions. Nonetheless, PCR has proved to be a powerful amplification tool to enhance the sensitivity of molecular diagnostic techniques.

Detection of PCR Products. The specific PCR amplification product containing the target nudeic add of interest is referred to as the amplicon. Because PCR produces an amplicon in substantial quantities, any of the basic methods previously described for detecting hybridization can be adopted for detecting spedfic am-plieons. Basically this involves using a labeled probe that is spedfic for the target sequence within the amplicon. Therefore, solution or solid-phase formats may be used with reporter molecules that generate radioactive, co-lorimetric, fluorometric, or chemiluminescent signals. Probe-based detection of amplicons serves two purposes: it allows visualization of the PCR product and it provides spedfidty by ensuring that the amplicon is the target sequence of interest and not the result of nonspecific amplification.

When the reliability of PCR for a particular amplicon has been well established, hybridization-based detection may not always be necessary. Confirming the presence of an amplicon of the expected size may be suffident. This is commonly accomplished by subjecting a portion of the PCR mixture, after amplification, to gel electrophoresis. After electrophoresis, die gel is stained with ethidium bromide to visualize the amplicon and, using molecular-weight-size markers, the presence of amplicons of appropriate size (i.e., the size of target sequence amplified depends on the primers selected for PCR) is confirmed (Figure 8-8).

Derivations of the PCR Method. The powerful amplification capadty of PCR has prompted the development of several modifications that enhance the utility of this methodology, particularly in the diagnostic setting. Specific examples indude multiplex PCR, nested PCR, quantitative PCR, RT-PCR, arbitrary primed PCR, and PCR for nucleotide sequencing.

Multiplex PCR is a method by which more than one primer pair is induded in the PCR mixture. This approach offers a couple of notable advantages. First, strategies that indude internal controls for PCR can be developed. For example, one primer pair can be directed at sequences present in all clinically rdevant

Size markers A in base pains

Figure 8-8 Use of ethidium bromide-stained agarose gels to determine the size of PCR amplicons for identification. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B, C, and D contain PCR amplicons typical of the enterococcal vancomycin-resistance genes vanAtfBI kb), vmiB (297 kb), and vanCl (822 kb), respectively.

Size markers in base pairs

Size markers in base pairs

/— Control amplicon (370 bp) — Mec A gene amplicon (310 bp)

Figure 8-9 Hihidram bromide-stained gels containing amplicons produced by multiplex PCR. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B and C show amplicons obtained with multiplex PCR consisting of control primers and primers specific for the staphylococcal methidllln-resisiance gene mecA. The presence of only the control amplicon (370 bp) in Lane B indicates that PCR was successful, but the strain on which the reaction was performed did noL contain mecA. Lane C shows both the control and the mecA (310 bp) amplicons, indicating that the reaction was successful and that the strain tested carries the mecA resistance gene.

/— Control amplicon (370 bp) — Mec A gene amplicon (310 bp)

Figure 8-8 Use of ethidium bromide-stained agarose gels to determine the size of PCR amplicons for identification. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B, C, and D contain PCR amplicons typical of the enterococcal vancomycin-resistance genes vanAtfBI kb), vmiB (297 kb), and vanCl (822 kb), respectively.

Figure 8-9 Hihidram bromide-stained gels containing amplicons produced by multiplex PCR. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B and C show amplicons obtained with multiplex PCR consisting of control primers and primers specific for the staphylococcal methidllln-resisiance gene mecA. The presence of only the control amplicon (370 bp) in Lane B indicates that PCR was successful, but the strain on which the reaction was performed did noL contain mecA. Lane C shows both the control and the mecA (310 bp) amplicons, indicating that the reaction was successful and that the strain tested carries the mecA resistance gene.

bacteria (Le„ the control or universal primers) and the second primer pair can be directed at a sequence spedfic for the particular gene of interest (i,e„ the test primers). The control amplicon should always be detectable after PCR, and its absence would indicate that PCR conditions were not met and the test would require repeating. When the control amplicon is detected, the absence of the test amplicon can be more confidently interpreted to indicate the absence of target nudeic add in the specimen rather than a failure of the PCR system (Figure 8-9).

Another advantage of multiplex PCR is the ability to search for different targets using one reaction. Primer pairs directed at sequences spedfic for different organisms or genes can be put together so that the use of multiple reaction vessels can be minimized. For example, multiplexed PCR assays containing primers to detect viral agents that cause meningitis or encephalitis (e.g., herpes simplex virus, enterovirus. West Nile virus) have been used in a single reaction tube. A limitation of multiplex PCR is that mixing different primers can cause some interference in die amplification process so that optimizing conditions can be difficult, especially as the number of different primer pairs used increases.

Nested PCR involves the sequential use of two primer sets. The first set is used to amplify a target sequence. The amplicon obtained is then used as the target sequence for a second amplification using primers internal to those of the first amplicon. The advantage of this approach is extreme sensitivity and confirmed specifidty without the need for using probes. Because production of the second amplicon requires the presence of the first amplicon, production of the second amplicon automatically verifies the accuracy of the first amplicon. The problem encountered with nested PCR is that the procedure requires open manipulations of amplified DNA that is readily, albeit inadvertently, aerosolized and capable of contaminating other reaction vials.

Arbitrary primed PCR uses short primers that are not specifically complementary to a particular sequence of a target DNA. Although they are not specifically directed, their short sequence (approximately 10 nudeotides) ensures that they will randomly anneal to multiple sites in a chromosomal sequence. On cycling, the multiple annealing sites result in the amplification of multiple fragments of different sizes. Theoretically, strains that have similar nudeotide sequences will have similar annealing sites and thus will produce amplified fragments (i.e., amplicons) of similar sizes. Therefore, by comparing fragment migration patterns following agarose gel electrophoresis, strains or isolates can be judged to be the same, similar, or unrelated.

Quantitative PCR is an approach that combines the power of PCR for the detection and identification of infectious agents with the ability to quantitate the actual number of targets originally in the clinical specimen. The ability to quantitate "infectious burden" has tremendous implications for studying and understanding the disease state (e.g., acquired immunodefidency syn drome [AIDS]), the prognosis of certain infections, and the effectiveness of antimicrobial therapy.

The PCR methods discussed thus far have focused on amplification of a DNA target. Reverse transcription PCR (RT-PCR) amplifies an RNA target. Because many clinically important viruses have genomes composed of RNA rather than DNA (e.g., human immunodeficiency virus [HIV], hepatitis B virus), the ability to amplify RNA greatly facilitates laboratory-based diagnostic testing for these infectious agents. The unique step to this procedure is the use of the enzyme reverse transcriptase that directs synthesis of DNA from the viral RNA template, usually within 30 minutes. Once the DNA has been produced, relatively routine PCR technology is applied to obtain amplification.

Real-Time PCR

Most conventional PCR-based tests used in clinical laboratories were developed in-bouse and needed to be performed in dedicated spaces to control or reduce cross contamination that was always a threat for producing false-positive results. Conventional PCR assays also require multiple manipulations including initial amplification of target nucleic acid, detection of amplified product by gel electrophoresis, and then confirmation by an alternative method such as Southern blotting or chemiluminescence techniques. In general, a conventional PCR assay would require a minimum of at least 4 to 6 hours from the time that extracted nucleic acid is placed into a thermal cycler to begin amplification to subsequent product detection.

Recently, small, automated instruments that combine target nucleic acid amplification with qualitative or quantitative measurement of amplified product have become commercially available. These instruments are noteworthy for four reasons: First, these new instruments combine thermocyding or target DNA amplification with the ability to detect amplified target by fluorescently labeled probes as the hybrids are formed (Le„ detection of amplicoo in real time). Second, because both amplification and product detection can be accomplished in one reaction vessel without ever opening, the major concern of cross contamination Of samples with amplified product assodated with conventional PCR assays is greatly lessened. Third, these instruments are not only able to measure amplified product (amplicon) as it is made, but because of this capability, they are also able to quantitate the amount of product and thereby determine the number of copies of target in the original specimen. And fourth, the amount of time to complete a real-time PCR assay is significantly decreased compared to conventional PCR-based assays because the time required for the post-PCR detection of amplified product is eliminated by the use of fluorescent probes. Also, some systems are able to perform rapid thermal cycling based on instrument design, detecting product in as little as 20 to 30 minutes.

Several instruments (also referred to as platforms) are available for amplification in conjunction with realtime detection of PCR-amplified products; examples are shown in Figure 8-10. Each instrument has unique features that permit some flexibility such that a clinical laboratory can fulfill its specific needs in terms of specimen capadty, number of targets simultaneously detected, detection format, and time for analysis (Table g-I). Nevertheless, all instruments have amplification (Le., thermal cycling) capability as well as an exdtation or light source, an emission detection source, and a computer interface to selectively monitor the formation of amplified product.

As with conventional PCR, nudeic add must first be extracted from the clinical specimen before real-time amplification. In prindple, real-time amplification is accomplished in the same manner as previously described for conventional PCR-based assays in which denaturation of double-stranded nucleic add followed by primer annealing and extension (elongation) are performed in one cyde, However, it is the detection process that discriminates real-time PCR from conventional PCR assays. In real-time PCR assays, accumulation of amplicon is monitored as it is generated. Monitoring of amplified target is made possible by the labeling of primers, oligonudeotide probes (oligoprobes) or amplicons with molecules capable of fluorescing. These labels produce a change in fluorescent signal that is measured by the instrument following their direct interaction with or hybridization to the amplicon. This signal is related to the amount of amplified product present during each cyde and increases as the amount of specific amplicon increases,

Currently, a range of fluorescent chemistries are used for amplicon detection; the more commonly used chemistries can be divided into two categories: (1) those that involve the nonspecific binding of a fluorescent dye (e.g., SYBER Green I) to double-stranded DNA and (2) fluorescent probes that bind spedfically to the target of interest. SYBER Green I chemistry is based on the binding of SYBER Green I to a site referred to as the DNA minor groove (where the strand backbones of DNA are doser together on one side of the helix than on the other) that is present in all double-stranded DNA molecules. Once bound, fluorescence of this dye increases more than 100-fold. Therefore, as the amount of double-stranded amplicon increases, the fluorescent signal or output increases proportionally and can be measured by the instrument during the elongation stage of amplification. However, a major disadvantage of this particular

Figure 8-10 Examples of real-time PCR instruments. A, Applied Biosystems. B, ¡Cycler. C, Light Cycler. D, SmartCycler. (A courtesy Applied Biosystems, Foster, Calif; B courtesy Bio-Rad Laboratories, Hercules, Calif; C courtesy Roche Applied Science, Indianapolis, Ind; D courtesy Cepheid, Sunnyvale, Calif.)

Figure 8-10 Examples of real-time PCR instruments. A, Applied Biosystems. B, ¡Cycler. C, Light Cycler. D, SmartCycler. (A courtesy Applied Biosystems, Foster, Calif; B courtesy Bio-Rad Laboratories, Hercules, Calif; C courtesy Roche Applied Science, Indianapolis, Ind; D courtesy Cepheid, Sunnyvale, Calif.)

means of detection is that it can detect both specific and nonspecific amplified products.

Three different chemistries commonly employed to detect amplicon in real time involve additional fluorescence-labeled oligonucleotides or probes. Sufficient amounts of fluorescence are only released either after cleavage of the probe (hydrolysis probes) or during hybridization of one (molecular beacon) or two oligonucleotides (hybridization probes) to the amplicon. These fluorescent chemistries for detection of amplified product are overviewed in Figure 8-11. Introduction of these additional probes increases the specificity of the PCR product. Also, there are real-time PCR instruments (e.g., Light Cycler, Roche Applied Science, Indianapolis,

Table 8-1 Examples of Commercially Available Real-Time Polymerase Chain Reaction (PCR) Instruments

Instrument*

Manufacturer

Detection Format

Turn-Around Timet

Ability to Detect Muttipie Targets

Maximum No. of Reactions per Run

Applied

Btosystems 7300 and 7500 Real-Time 'CR Systems

Applied Biosystems Foster, Calif

Hydrolysis probe (TaqMan) or molecular beacons

40min-approx 2 hr

Yes

96*

ICyclerlQ Real-Time PCR Detection System

Bio-Rad Laboratories Hercules, Calif

Hydrolysis probe (TaqMan), hybridization probes (FRET), or molecular beacons

30 mln-1.5 hr

Yes

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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