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Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
SUMMARY INTRODUCTION REAL-TIME PCR INSTRUMENTS REAL-TIME PCR PROBE TECHNOLOGIES 5' Nuclease (TaqMan) Probes Molecular Beacons FRET Hybridization Probes NUCLEIC ACID EXTRACTION Manual Extraction Automated Extraction Auxiliary Procedures To Enhance Extraction REAL-TIME PCR ASSAY DEVELOPMENT Target Nucleic Acid Selection PCR Primer and Probe Design Assay Optimization BIOSAFETY CONSIDERATIONS QUALITY CONTROL AND QUALITY ASSURANCE Verification and Validation Positive and Negative Controls Internal and Inhibition Controls Reagents Quality Assurance Contamination IMPLEMENTATION OF REAL-TIME PCR TESTING IN THE CLINICAL MICROBIOLOGY LABORATORY Facilities Requirements Personnel Requirements Work Flow Design Example of work flow design: Example of work flow design: COSTS Royalties Reagents and Instrumentation Personnel Cost Savings at the Bedside Coding and Reimbursement APPLICATION OF REAL-TIME PCR FOR CLINICAL MICROBIOLOGY TESTING BACTERIA OTHER THAN MYCOBACTERIA SPP. General Bacteria Slow-Growing or Poorly Culturable Bacteria Agents of Community-Acquired Pneumonia Agents of Meningitis Potential Agents of Bioterrorism Bacterial Antibiotic Resistance Genes MYCOBACTERIA VIRUSES Qualitative Viral Assays Herpes simplex virus. Herpes simplex virus CNS disease. Herpes simplex virus dermal and genital disease. Varicella-zoster virus dermal disease. Varicella-zoster virus CNS disease. Cytomegalovirus CNS disease. Epstein-Barr virus CNS lymphoproliferative disease. Enterovirus CNS disease. Polyomaviruses. JCV CNS disease. Parvovirus. West Nile virus. Respiratory Viruses Influenza viruses. Rous sarcoma virus. Adenovirus. Metapneumovirus. Parainfluenza virus. Severe acute respiratory syndrome coronavirus. Poxviruses QUANTITATIVE VIRAL ASSAYS Cytomegalovirus Epstein-Barr Virus BK Virus Viral Hepatitis Agents Human Immunodeficiency Virus FUNGI Aspergillus Species Candida Species Pneumocystis jiroveci PARASITES Plasmodium spp. Babesia spp. Trypanosoma spp. Leishmania spp. Toxoplasma spp. Trichomonas spp. Cryptosporidium, Entamoeba, and Giardia spp. ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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Real-time PCR testing platforms provide equivalent sensitivity and specificity as conventional PCR combined with Southern blot analysis. Since the nucleic acid amplification and detection steps are performed in the same closed vessel, the risk for release of amplified nucleic acids into the environment, and contamination of subsequent analyses, is negligent compared with conventional PCR methods. Real-time PCR instrumentation requires considerably less hands-on time and testing is much simpler to perform than conventional PCR methods. Additionally, accelerated PCR thermocycling and detection of amplified product permits the provision of a test result much sooner for real-time PCR than for conventional PCR. The combination of excellent sensitivity and specificity, low contamination risk, ease of performance and speed, has made real-time PCR technology an appealing alternative to conventional culture-based or immunoassay-based testing methods used in the clinical microbiology for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.
| REAL-TIME PCR INSTRUMENTS |
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96-microwell format) instruments, which
include the ABI Prism series (7000, 7300, and 7500), the MyiQ
and iCycler, Mx4000, MX3000p, Chromo4, Opticon and Opticon 2, and
SynChron, may be particularly useful in laboratories with
large numbers of specimens. However, thermocycling on these instruments
is slower than on other lower capacity instruments, including the
LightCycler 1.0, LightCycler 2.0, and SmartCycler II. This is due to
the use of a solid-phase material for heat conductance (heating block
principle). The large-capacity instruments support high-volume testing
while the rapid, lower capacity instruments permit the work flow
flexibility that may be especially useful for laboratories that test
fewer samples. In summary, work load and work flow issues may dictate
which system is best for different-sized laboratories and test
volumes.
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All the instruments support all or some of the dyes used for TaqMan probes and molecular beacons. Currently, only the LightCycler supports fluorescence resonance energy transfer (FRET) hybridization probe detection with melting curve analysis. Quantitation of target nucleic acid is possible with any of the instruments and supported detection formats.
Recently, analyte-specific reagents (ASRs) and Food and Drug Administration (FDA)-approved kits have become available in the United States for testing on several real-time PCR instruments. The commercial availability of these reagents now make it considerably easier for many clinical microbiology laboratories to adapt real-time PCR testing platforms into their work flow. Because laboratory-developed (also referred to as in-house developed or home brew) real-time PCR tests required considerable expertise to develop and validate, they are generally limited to larger laboratories, especially referral laboratories. The availability of ASRs and kits will also facilitate the development of common testing protocols and standards so that proper comparative clinical studies can be performed and ultimately reliable test results can be ensured for the patient.
In addition to the usual considerations for new instrument purchase (physical space requirement, cost of instrument, disposables, and reagents, instrument maintenance and service, reliability, upgrades, etc.), selection of a real-time PCR instrument and real-time detection format requires consideration of test volume, probe detection requirements, turnaround time for results, personnel requirements, and software. Also, sample preparation requirements must be considered as this can add to the hands-on time per sample, turnaround time, and expense. Several manufacturers have developed semiautomated nucleic acid extraction instruments for use in tandem with real-time PCR instruments. These include the MagNA Pure LC and MagNA Pure Compact for use with the LightCycler instrument, the GeneExpert for use with the SmartCycler II, and the ABI Prism 6700 for use with the ABI Prism series instruments.
| REAL-TIME PCR PROBE TECHNOLOGIES |
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Sensitive and specific detection is possible with real-time PCR by using novel fluorescent probe technology probes. Three types of nucleic acid detection methods have been used most frequently with real-time PCR testing platforms in clinical microbiology: 5' nuclease (TaqMan probes), molecular beacons, and FRET hybridization probes (Fig. 1). These detection methods all rely on the transfer of light energy between two adjacent dye molecules, a process referred to as fluorescence resonance energy transfer (500). Collectively, these three types of probes are frequently referred to as FRET probes and this general term has been used in some sections of this review. However, when specifically referring to each of these three types of probes, only FRET appears in the name of one, i.e., FRET hybridization probes. Because FRET hybridization probes consist of two separate probes, the term dual FRET hybridization probes has also been used to describe this specific type of nucleic acid detection method.
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A single TaqMan probe can be used for detection of amplified target DNA. If the intent of the assay is to differentiate a single nucleotide polymorphism from a wild type sequence in the target DNA, then a second probe with the complementary nucleotide(s) to the polymorphism and a fluorescent dye with a different emission spectrum is utilized. Thus, TaqMan probes can be used to detect a specific, predefined polymorphism under the probe in the PCR amplification product. For this application, two reaction vessels are required, one with a complementary probe to detect wild-type target DNA and another for detection of a specific nucleic acid sequence of a mutant strain. Because TaqMan probes require 60°C for efficient 5' nuclease activity, the PCR is usually cycled between 95 and 60°C for amplification. In addition, the cleaved (free) fluorescent dye accumulates after each PCR temperature cycle, and therefore can be measured at any time during the PCR cycling, including the hybridization step. This is in contrast to molecular beacons and FRET hybridization probes, for which fluorescence can only be measured during the hybridization step.
Typically, a single molecular beacon is used for detection of a PCR amplification product and multiple beacon probes with different reporter dyes are used for single nucleotide polymorphism detection. By selection of appropriate PCR temperatures and/or extension of the probe length, molecular beacons will bind to the target PCR product when an unknown nucleotide polymorphism is present but at a slight cost of reduced specificity. There is not a specific temperature thermocycling requirement for molecular beacons, so temperature optimization of the PCR is simplified.
FRET hybridization probe technology permits melting curve analysis of the amplification product. If the temperature is slowly raised, eventually the probes will no longer be able to anneal to the target PCR product and the FRET signal will be lost. The temperature at which half the FRET signal is lost is referred to as the melting temperature of the probe system. The Tm depends on the guanine plus cytosine content and oligonucleotide length. In contrast to TaqMan probes, a single nucleotide polymorphism in the target DNA under a hybridization FRET probe will still generate a signal, but the melting curve will display a lower Tm. The lowered Tm can be characteristic for a specific polymorphism underneath the probes; however, a lowered Tm can also be the result of any sequence difference under the probes. The target PCR product is detected and the altered Tm informs the user there is a difference in the sequence being detected. Generally, more than three base pair differences under a FRET hybridization probe prevent hybridization at typical annealing temperatures and are not detected.
This trait of FRET hybridization probes is advantageous in cases where the genome of the organism is known to mutate at a high frequency, such as with viruses. When a single or limited number (<3) of known polymorphisms occur between two similar targets, FRET hybridization probes can also be used for discriminating strains of organisms. An example of this application is the identification of herpes simplex virus type 1 (HSV-1) and HSV-2 (Fig. 2). Like molecular beacons, there is not a specific thermocycling temperature requirement for FRET hybridization probes. Molecular beacons and FRET hybridization probes, unlike TaqMan probes, are both recycled (conserved) in each round of PCR temperature cycle. Also, for Molecular beacons and FRET hybridization probes, unlike TaqMan probes, fluorescent signal does not accumulate as PCR product accumulates after each PCR cycle.
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| NUCLEIC ACID EXTRACTION |
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A few general comments about extraction of nucleic acid from microorganisms can be made. The thick cell wall of gram-positive bacteria is more difficult to disrupt than the relatively thinner cell wall of gram-negative bacteria. Substances that may inhibit amplification such as heme in blood or bile in stool must be removed. The released nucleic acids should be maintained in an aqueous solution to protect them from degradation. Nucleic acids should be eluted into a small volume in order to maximize detection.
Extraction of clinical specimens can be accomplished either by manual or automated methods. A survey of the literature demonstrates the ability of various commercially available methods to successfully extract a wide variety of specimens for bacterial, viral, and fungal targets (Tables 2 and 3).
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Processing of samples by manual methods requires multiple manipulations. As the number of samples to be extracted increases, so does the potential for contamination due to increased manipulation. In the United States, Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations (http://www.cms.hhs.gov/clia/) consider manual extraction high-complexity testing, and therefore this type of testing can only be performed by laboratory personnel with appropriate academic credentials. In order to ensure reproducible results, extensive training is necessary to achieve consistency among laboratory personnel performing manual extraction. Some manual kits use ethanol to precipitate the nucleic acids. If not properly removed, excess ethanol residues can inhibit the PCR (502). Finally, manual extraction is a laborious, time-consuming process which requires the undivided attention of the technologist performing this technique in order to ensure optimal results.
Automated extraction systems have certain inherent advantages over manual methods. Recovery of nucleic acids from automated instruments is consistent and reproducible. Automated extraction systems keep sample manipulation to a minimum, reducing the risk for cross contamination of samples. Many of the instruments are closed systems, further reducing the risk for contamination. Automated systems are typically walk-away systems, and do not require constant attention, which permits personnel to perform other duties. The procedures associated with these instruments could potentially be classified as moderate complexity based on the the Clinical Laboratory Improvement Amendments of 1988 (13). Therefore, laboratory assistants may be able to perform sample extraction with these instruments. Finally once these systems have been validated and proper maintenance procedures are in place, quality control monitoring is less intensive than that required for manual extraction (137).
While the benefits of automated extraction are considerable, there are potential drawbacks. It is most economical when instruments are fully loaded; therefore, a significant number of samples (50 to 100/day) should be processed in order to justify the capital investment that is required for these instruments. The footprints of automated extraction instrumentation may require space that is not currently available in the laboratory. In addition to the cost for equipment, costs for disposables also need to be considered. Some vendors are now manufacturing smaller versions of earlier models of their instruments (Table 3). While these instruments extract significantly fewer samples at a time, they are less expensive and have a smaller footprint than the parent instrument. These smaller versions may be viable options for smaller laboratories which process lower numbers of specimens.
The swab extraction tube system (S.E.T.S.; Roche Diagnostics Corporation) kit, shown in Fig. 3, is a simple method for rapidly and efficiently recovering specimen attached to and absorbed into the fibers of a collection swab. For some organisms, studies have demonstrated that specimen which is retrieved in a microcentrifuge tube by the S.E.T.S. method, can be directly, or after a quick lysis step (boiling), analyzed by a LightCycler real-time PCR instrument (195, 499). Alternatively the centrifuged material can be extracted by the MagNA Pure instrument to obtain a cleaner preparation of nucleic acids.
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| REAL-TIME PCR ASSAY DEVELOPMENT |
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For real-time PCR a few key components should be optimized in order to achieve maximum results (17, 35, 228, 483). These factors include magnesium concentration, which allows the polymerase enzyme to function at an optimal level; primer and probe concentrations, which affect the sensitivity and specificity of the assay respectively; and the use of additives such as dimethyl sulfoxide, which can aid in the denaturation of nucleic acids with high G+C. The type of polymerase enzyme utilized can also play a significant role, polymerases which permit hot-start PCR are preferrable. These enzymes do not function until a critical maximum temperature is reached, which reduces the generation of nonspecific sequence fragments.
| BIOSAFETY CONSIDERATIONS |
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The MagNA Pure mixes a guanidinium isothiocyanate-containing lysis solution with the sample and incubates it at room temperature for 2 min. We have found (unpublished observations) that this treatment renders 108 Staphylococcus aureus/ml nonviable. However, further studies are required to determine if guanidinium has the same effect on other infectious agents. Until these studies are completed individuals using real-time PCR in clinical laboratories should practice universal precautions, i.e., treating all specimens as if they were infectious (10).
In the past, infectious agents, such as anthrax, have been weaponized for use in biological warfare. The intentional release of anthrax spores in the U.S. mail system in the fall of 2001 emphasized the urgent need for rapid and safe laboratory techniques for detecting Bacillus anthracis in suspicious powders as well as human specimens (179, 289, 350). The Centers for Disease Control and Prevention (CDC) has issued guidelines for the processing and testing of specimens obtained from a suspected outbreak of bioterrorism, in order to protect first-line workers (direct healthcare providers and laboratory workers) (10). In the case of a smallpox outbreak, rapid and accurate laboratory detection is critical in order to quickly contain the infection, however, this may be difficult as smallpox is a level 4 organism, and as such, must be tested at institutions with specialized biosafety level 4 containment facilities (i.e., CDC or United States Army Medical Research Institute of Infectious Diseases).
Autoclaving has been shown to be an effective way to inactivate potential agents of bioterrorism, while permitting the nucleic acid to remain intact for analysis by PCR assays (119, 125, 179, 289, 350). The authors of these publications demonstrated that autoclaving anthrax spores and vaccinia virus, a close relative of smallpox virus, destroyed their ability to be infectious, while not affecting the integrity of their nucleic acid so it could be detected by PCR techniques.
As indicated in the preceding discussion, S.T.A.R. buffer (Roche Diagnostics Corporation) not only stabilizes nucleic acid during transport at room temperatures, but can inactivate pathogens. We have observed that S.T.A.R. buffer can inactivate many bacteria, including such pathogens as Mycobacterium tuberculosis and Escherichia coli OH157:H7 isolated from culture, or present in complex matrices such as respiratory and stool specimens without damaging the integrity of the DNA (unpublished data). The pathogen-inactivating properties of S.T.A.R. buffer provides laboratories an added level of safety for processing and transporting pathogens for nucleic acid analysis.
| QUALITY CONTROL AND QUALITY ASSURANCE |
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The most recent document addressing quality control standards for molecular test systems is the revised CLIA 1988 document published in the Federal Register, 24 January 2003 (4). This document addresses requirements for certain quality control provisions and personnel qualifications. It combines and reorganizes requirements for test management, quality control and quality assurance, and also changes the requisite consensus for grading proficiency testing challenges. The CLIA 88 document stipulates that prior to test implementation, clinical laboratories verify the manufacturer's performance specifications and confirm they can be replicated by laboratory personnel when following the procedure. For laboratory developed tests or modification of test systems, laboratories are required to establish their own performance specifications prior to implementation of the new or modified test.
Because nucleic acid test methods are changing and evolving so rapidly, existing guidelines have been difficult to apply. The challenges to clinical laboratories include determining the type of verification experiments required for a real-time PCR assay and an acceptable number and type of specimens to evaluate. Providing a single set of guidelines for real-time PCR which envelops all the necessary verification and validation by all accreditation agencies would be of great benefit to laboratories acquiring this new technology. Along with the need for a well defined quality control program for real-time PCR qualitative assays, there is need for guidelines for quantitative real-time PCR assays. To date such guidelines only exist for a select number of blood-borne viruses (341).
Quality control allows the laboratory to minimize the reporting of inaccurate results, to report results with a high degree of confidence and to decrease costs by detecting errors prior to reporting results (137). One goal of the laboratory quality control program is to reduce the number of controls needed for reporting acceptable results. The following information relates to specific controls used during testing as well as the quality control of reagents used for testing. This discussion is not intended to be all-inclusive nor definitive, and is based to some extent on experience at Mayo Clinic with real-time PCR and our interpretation of published guidelines for generic molecular testing.
A blank control such as water or buffer is often used as a negative control. However, an optimal negative control is a sample containing nontarget nucleic acid to demonstrate that nonspecific PCR amplification and detection of amplified product is not occurring. In addition, the negative control is used to demonstrate that the reagents are not contaminated with target nucleic acid and can be used to compensate for background signal generated by the reagents. The recommendation for a negative control every fifth tube to monitor PCR contamination (332) may be excessive with real-time PCR assays. The closed system for amplification and detection used with real-time PCR virtually eliminates the amplicon contamination caused by the opening and closing of reaction vessels which is problematic with conventional PCR and detection methods. Even with the closed system of real-time PCR, the laboratory may still choose to add uracil-N-glycosylase to the PCR mix for another level of amplicon contamination control.
The 2003 revisions to CLIA 88 rule does not specifically address real-time PCR assays but recommendations from the molecular testing sections can be applied to real-time PCR assays. Table 5 summarizes the 2003 revised CLIA 88 document (4) and CLSI (332) quality control recommendations. In both of these documents it is recommended that each molecular amplification run of samples contain positive and negative controls. Additionally, in the CLIA document it is indicated that a test system which includes nucleic extraction also include a positive and negative control, with the positive control capable of detecting errors in the nucleic acid extraction procedure. For a quantitative assay, two positive controls representing two different concentrations of target nucleic acid are recommended in both the CLIA 88 and CLSI documents. Laboratories should establish the number and frequency of controls based on manufacturers criteria and agency recommendations.
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Based on the requirements of regulatory or accrediting agencies, individual laboratories should determine when an internal control is required in an assay. For example, the 2003 revision to CLIA 88 document does not address internal controls nor have a requirement for assessment of inhibition of PCR chemistry. In contrast, the College of American Pathologist (CAP) molecular pathology inspection checklist indicates that the laboratory must determine the likelihood of the generated result being a false-negative result due to inhibition when there is no amplification of product (76). If the test is performed according to manufacturer instructions, published data containing the inhibition rate may be used for documentation. Internal controls for laboratory developed assays or modified FDA assays should be determined on a case-by-case basis taking into account the probability that the specimen source contains inhibitory substances. Specimen types such as stool or sputum are generally more inhibitory to PCR chemistry than serum or plasma specimens. Also, the assay performance characteristics (sensitivity, specificity, accuracy, etc.), the implications of a false-negative result and the degree to which a clinical diagnosis depends on laboratory results, require consideration. Internal controls may be naturally present in the original specimen, added to the specimen prior to extraction, or added to the PCR reagent mix before amplification. The simplest way to establish inhibition is to add target nucleic acid to a portion of the sample and perform the test to show that if target nucleic acid were present, the PCR would have been positive. Unfortunately, this approach increases the cost of the assay.
Examples of the different types of internal controls that have been used for real-time PCR assays are shown in Table 6. Homologous and heterologous internal controls are those which do not naturally occur within the specimen source. These have also been referred to as exogenous controls as they must be added to the specimen. Homologous controls are coamplified with target DNA using the same PCR primers. However, the internal sequence of the homologous control DNA internal to the PCR primer sites is genetically engineered to be different from the target DNA such that a different product signal occurs with FRET detection.
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Whether PCR primers are purchased from vendors or laboratory developed, some method of chromatographic purification should be applied. Purification recovers oligonucleotides of the correct length. Truncated oligonucleotides can affect a PCR by consuming reaction reagents and forming nonspecific amplification products (159). The presence of these irregular oligonucleotides can also falsely elevate the final concentration of the working primers thus affecting the performance of the assay. The CLSI MM3 guideline recommends that laboratories obtain a certificate of analysis from PCR primer vendors. These certificates may contain sequence data, base composition, molecular weight of the sequence, concentration and method of purification (332). Vendors may provide PCR primer concentration, but these concentrations should be verified in the laboratory. Laboratories synthesizing their own oligonucleotide PCR primers should perform chromatographic purification and determine also PCR primer concentration. In compliance with the CAP Molecular Pathology inspection checklist, each new lot of reagent should be tested in parallel with the old reagent lot using both positive and negative patient samples ensuring the same results are obtained with both reagent lots (76).
Purification of FRET probes is especially important to separate both dye and oligonucleotides that have not coupled to form the FRET probes and to remove oligonucleotides with an incorrect length (554). While the quality of probe synthesis has improved greatly over the past few years, quality control is required to avoid probe lots with reduced performance. The CLSI MM3 guideline for PCR primers discussed above should also apply to FRET probes. Some vendors provide a tracing (chromatogram, polyacrylamide gel electrophoresis analysis, etc.) of the purified FRET probe as part of their quality control documentation which may augment quality control. Another quality control service provided at a nominal charge by some vendors is to determine if a particular FRET hybridization probe set is capable of producing FRET. The company will design and synthesize an oligonucleotide complementary to both probes and a melting curve analysis is performed. The production of a melting peak at the predicted Tm will confirm that the FRET hybridization probe set is capable of producing FRET and is acceptable to use. This probe validation process can also be completed in the laboratory by following the method provided on the Idaho Technology website (http://www.idahotech.com) under probe classroom.
Proper storage of reagents can result in an increase in shelf life. FRET probes may arrive in a lyophilized form and the recommendation is to store them at room temperature until resuspended. Some manufacturers state that the hydrated probes should be stored at 4°C for daily use or aliquoted into smaller volumes and stored at –20°C. Numerous freeze-thaw cycles can be detrimental to the FRET probes. The PCR primers can be stored in a similar manner as the probes. The completed master mix (containing primers and probes) can be stored at –20°C for extended periods of time, without degradation of the mix (452). We have also found this to be true with most of our real-time PCR assay master mix reagents used at the Mayo Clinic. The complete mix is stored at either 4°C or –20°C (assay dependent) for 1 to 6 months without loss of activity. However, we have observed that the length and temperature of storage are assay dependent and conditions of storage require validation for each assay. The advantage of freezing the master mix is assay reproducibility, time savings in setting up assays, and reduced reagent contamination (452).
Certain types of sample sources are known to contain a high concentration of organisms that may lead to specimen-to-specimen contamination, namely, viral agents. The inclusion of negative controls and continual trend analysis of the assay are used to recognize a contamination event. Additionally, unidirectional work flow should be followed. Separation of procedural steps will require separate work spaces in the laboratory, as detailed below under the ssection on facilities requirements. As with all methods performed in the laboratory, good laboratory practice is critical for accurate results.
| IMPLEMENTATION OF REAL-TIME PCR TESTING IN THE CLINICAL MICROBIOLOGY LABORATORY |
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The reagent preparation area should be kept free of all patient specimens and DNA extracts. Protocols for the sample preparation area should minimize the number of tubes that are simultaneously open. Each of the work areas should contain dedicated working materials, reagents, and pipetting devices. Reagents should be prepared and aliquoted into single use or small volumes. This ensures ease of use and less chance for contamination.
All working surfaces should be cleaned before and after use, preferably with a reagent that destroys nucleic acid such as a 5% bleach solution. The manufacturer's recommendations should be followed for cleaning of instrument components (e.g., carousels with the LightCycler), processing blocks, and other instrument surfaces and parts.
Gloves should be changed frequently, at least before beginning each of the separate tasks required in a dedicated work area and should always be changed if moving from one work area to another work area. The use of aerosol-resistant pipette tips and pipette tips long enough to prevent specmien contact with the pipetter aids in the prevention of specimen contamination (502). Enzyme contamination control systems such as uracil-N-glycosylase can be incorporated into the real-time PCR master mix as an added safeguard to sterilize amplified product that may be carried over to subsequent batches of tests.
Clinical microbiologists who have not had formal training in molecular microbiology still possess many of the critical skills necessary for success in performing real-time PCR testing. Especially important is meticulous attention to detail, strict adherence to standard operating procedures, and use of aseptic technique.
These skills are easily transferable from culture based conventional microbiologic testing to real-time PCR testing. At the Mayo Clinic we noted that providing training on the basics of accurate pipetting was fundamental, especially for those lacking experience with micropipetting.
Well-written training materials, including training checklists and detailed standard operating procedures for each real-time PCR test, should be available. The training checklist serves to standardize the training of all personnel. At the Mayo Clinic, we believe that identifying a technical expert to provide one-on-one training for real-time PCR is critical. Technologists are required to successfully complete a panel of unknown samples and perform the procedure under direct observation of the technical expert to ensure flawless manipulations throughout the procedure. They also are required to analyze a previous run of samples with a variety of unusual results. This allows them to perfect their skills manipulating the computer software associated with the real-time PCR instrument and ensures consistent analysis and reporting of results. Overall, our technologists have been very enthusiastic about implementation of real-time PCR and excited to learn the new technology.
The availability of resources for troubleshooting is a consideration when selecting a molecular platform for the clinical laboratory. Laboratory-developed tests require that the technical resources to resolve problems related to the assay are available within the laboratory. Use of ASRs and United States Food and Drug Administration-approved tests allow the use of technical support resources available from the manufacturer for troubleshooting problems related to the assay or instrumentation.
Not unlike other microbiology tests, work flow and testing schedules for real-time PCR tests are determined by the arrival times of specimens into the laboratory, clinical urgency for the results, and laboratory hours of operation. Many of the real-time PCR platforms are most efficiently run in a batch mode. Some vendors provide identical thermocycling protocols for different ASRs for the same instrument. This allows testing for multiple analytes within the same run, which enhances the efficiency of the testing platform.
Example of work flow design: real-time PCR for detection of group A streptococci from throat swabs. At the Mayo Clinic in August 2002 we replaced a conventional testing method (rapid antigen screen with backup culture for rapid antigen negative results) with the LightCycler Strep-A assay (Roche Diagnostics Corporation, Indianapolis, IN) (456, 499). This real-time PCR test is as sensitive as the gold standard method, culture, and provides same-day conclusive results for all patients, whereas the antigen/backup culture method required up to 48 h for a conclusive result for the majority of patients. A simple lysis and extraction method of the swab sample is performed using the S.E.T.S. tube (Roche Diagnostics Corporation) before testing in the LightCycler (Fig. 5).
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30%,
85% of patients had to wait up to 48 h for a
conclusive result by culture. This was because with 30%
true-positive cases and 70% true-negative cases, half of the
true-positive cases, or 15%, were detected by rapid antigen and the
other half, or 15% of true-positives, as well as the 70% true-negatives
required detection by culture. Cultures are held for 48
h. In discussions with our health care providers, it became clear that there were other considerations beyond education on test performance. These included, specimen transportation and arrival times, (especially for samples coming from more distant clinics) and issues related to the expeditious provision of antibiotics (i.e., closing times of local pharmacies). Based on these considerations, we implemented testing five times daily (4:00 a.m., 11:30 a.m., 3:00 p.m., 5:30 p.m., and 8:30 p.m.), 7 days/week, with additional batches set up as needed during the peak season.
Additionally, we have eliminated most of the follow-up procedures required by health care providers by using the following innovative processes with our clinical colleagues (456). Patients identify their preferred pharmacy at the time the throat swab is collected. A standardized prescription form (including the antibiotics prescribed and the patient's preferred pharmacy) is completed by the healthcare provider, which then accompanies the specimen to the laboratory. At the conclusion of the test run, results are entered into the laboratory information system and transmitted to the patient's electronic medical record. The results from the electronic medical record are delivered to a computerized message center, allowing patients to obtain their secure results by telephone and pick up the prescription prepared for them at their selected pharmacy. Prescriptions are faxed to numerous local and regional pharmacies by the clinical microbiology laboratory staff (Fig. 5).
In a comparison of the personnel required for performing rapid antigen test and back up culture of antigen negative specimens, we realized a savings of 2.1 full time equivalents. This is based on an annual testing volume of 26,000 tests with the rapid antigen test being performed at four satellite locations in Rochester, Minnesota. Performance of the Roche Strep-A ASR test allowed us to centralize testing in one location. This results in a more efficient process that saved personnel effort.
In summary, the introduction of this rapid real-time PCR assay for detection of streptococcal pharyngitis has streamlined both the testing procedure and resulted in significant personnel savings in the laboratory. Most importantly, we have also implemented new procedures in tandem with this technology that facilitate the expeditious provision of appropriate antimicrobial therapy for our patients.
Example of work flow design: real-time PCR for detection of herpes simplex and varicella-zoster infections. In contrast to PCR testing for group A streptococci, the support of our clinical colleagues implementing this test was less of an issue. This was due in part to the fact that conventional PCR had been used for a number of years at our institution for detection of HSV in spinal fluid as well as other viruses such as hepatis C virus, and human immunodeficiency virus.
We currently use commercially available ASRs for both varicella-zoster virus (Roche Diagnostics Corporation) and HSV (Roche Diagnostics Corporation) testing with the LightCycler instrument. Testing is performed three to six times daily 6 days a week. Nucleic acid is extracted from the specimen using the automated MagNA Pure system (Roche Diagnostics Corporation) by laboratory assistants in our initial processing area. Real-time PCR testing is performed and results entered into the laboratory information system and transmitted to the patient's electronic medical record. In comparison to viral culture, which may take 14 days or longer to complete, the majority of results are available the same day the specimen is received (118). For these real-time PCR virology assays, efficiencies were gained in faster turnaround time and improved sensitivity compared to culture. Downstream, this can lead to a decrease in the number of tests re
