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Journal of Clinical Microbiology, January 1998, p. 191-197, Vol. 36, No. 1
Roche Molecular Systems, Branchburg, New
Jersey 08876
Received 26 June 1997/Returned for modification 10 September
1997/Accepted 10 October 1997
We constructed internal controls (ICs) to provide assurance that
clinical specimens are successfully amplified and detected. The IC
nucleic acids contain primer binding regions identical to those of the
target sequence and contain a unique probe binding region that
differentiates the IC from amplified target nucleic acid. Because only
20 copies of the IC are introduced into each test sample, a positive IC
signal indicates that amplification was sufficient to generate a
positive signal from targets present at the limit of test sensitivity.
The COBAS AMPLICOR Chlamydia trachomatis, Neisseria
gonorrhoeae, Mycobacterium tuberculosis, and human
hepatitis C virus tests exhibited inhibition rates ranging from 5 to
9%. Approximately 64% of these inhibitory specimens were not
inhibitory when a second aliquot was tested. Because repeatedly
inhibitory specimens were not reported as false negative and because
additional infected specimens were detected during retesting, test
sensitivities were 1 to 6% greater than they would have been if the IC
had not been used.
Because of its ability to
specifically amplify minute quantities of nucleic acid, PCR has been
applied with great success in clinical diagnostics (7, 8,
29). Relatively simple procedures for extracting nucleic acids
from clinical specimens provide samples of reasonable purity without
requiring hazardous chemicals and extensive manipulation
(10). Nevertheless, extracted clinical specimens may contain
impurities that inhibit enzyme-based nucleic acid amplification
processes. Numerous studies have demonstrated that a small, but
significant, proportion of clinical specimens contain substances that
inhibit PCR, the ligase chain reaction (LCR), and
transcription-mediated amplification (TMA) (1-6, 11, 14, 21, 22,
24-26, 30).
Unless inhibitory specimens are identified, negative amplification test
results do not necessarily indicate absence of infection. Inhibitory
specimens can be identified by monitoring amplification of a second
target nucleic acid, which serves as an internal control (IC).
Obtaining a positive signal from the second target demonstrates successful amplification, thereby validating a negative result for the
primary target.
A normal cellular gene sequence, which is expected to be present in all
specimens, can be used as an IC (13, 20). This approach has
the advantage of monitoring the integrity of the nucleic acid target;
in improperly collected, stored, or processed specimens, the endogenous
target will be absent (or degraded) and fail to yield a positive
result. A disadvantage is that endogenous sequences may not accurately
reflect amplification of the primary target due to differences in the
primer sequences, size of the amplified product, and the relative
amounts of the two targets.
There is another approach to monitoring amplification, using a
synthetic IC as a proxy for the primary target, that overcomes the
inherent limitations of an endogenous IC. One design of a synthetic IC
is a plasmid DNA or an in vitro RNA transcript with primer binding
regions identical to those of the target sequence, a randomized
internal sequence similar to the target sequence in length and base
composition, and a unique probe binding region that differentiates the
IC from amplified target nucleic acid (27). These features
ensure equivalent amplification of the IC and the target nucleic acid.
A limited number of IC molecules are added to each test sample and
coamplified with target nucleic acid; thus, a positive IC signal
assures amplification sufficient to generate a positive signal from
very small quantities of target. When introduced into the amplification
reaction mixture, the IC can monitor amplification and detection. When
introduced into the unprocessed specimen, the IC can also monitor
nucleic acid recovery during specimen preparation. Unlike a
housekeeping gene, a synthetic IC cannot be used to monitor the
integrity of the target nucleic acid in the specimen.
Here, we describe the properties of the IC used in COBAS AMPLICOR and
AMPLICOR tests and explain how to use and interpret the IC results
during routine clinical testing. We also demonstrate the clinical
utility of using an IC: increased sensitivity is achieved because
reporting false-negative results is avoided and because additional
positive results are detected by retesting inhibitory specimens.
Construction of ICs.
A unique IC was constructed for each
test by inserting the test-specific IC DNA sequence into a DNA plasmid.
All ICs contain the same probe binding sequence, which makes it
possible to detect all ICs with a single probe. The probe binding
sequence was designed to minimize homology with primers and target
sequences.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
An Internal Control for Routine Diagnostic PCR:
Design, Properties, and Effect on Clinical Performance
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Preparation of ICs. Cells from cultures of plasmid-containing bacteria grown overnight were harvested and extracted by a standard alkaline lysis method (23). DNA was recovered from the lysate with isopropanol (23) and purified by CsCl2 density gradient centrifugation. The purified plasmid DNA was then linearized with an appropriate restriction endonuclease and purified by phenol-chloroform extraction.
(i) ICs for C. trachomatis-N. gonorrhoeae and M. tuberculosis tests. The C. trachomatis-N. gonorrhoeae and M. tuberculosis ICs are supplied as stock solutions. Prior to use, 0.1 ml of IC stock solution is added to 1.7 ml of master mix to yield an amplification-ready working master mix containing 20 copies of IC plasmid per 50 µl. These IC stock solutions were prepared by serial dilution of purified plasmid DNA in a Tris-buffered solution containing EDTA, poly(A) RNA, sodium azide, and the dye amaranth (Sigma); amaranth was included to enable visual confirmation that IC was added to master mix.
(ii) IC for HCV test. The HCV IC was generated by transcribing HCV plasmid DNA, using the MEGAscript (Ambion, Inc.) in vitro transcription kit. Purified plasmid DNA was linearized with EcoRI restriction endonuclease and transcribed to yield 352-nucleotide-long RNA transcripts. The transcripts contained the 229-nucleotide IC sequence, 6 upstream nucleotides (the transcription start site), 14 downstream nucleotides, 30 adenylate nucleotides, and 3 nucleotides of the EcoRI recognition sequence.
The transcripts were purified from plasmid DNA by digesting the transcription reaction mixture with RNase-free DNase, followed by extraction with phenol-chloroform. The full RNA transcripts were then separated from unincorporated ribonucleotides and truncated transcripts by oligo(dT) cellulose affinity chromatography and were serially diluted in a buffered solution containing EDTA, poly(A) RNA, and sodium azide to yield an IC stock solution. Prior to use, 50 µl of the stock solution is added to 14 ml of HCV specimen diluent. Diluting processed specimens with specimen diluent (see "Specimen collection and processing" below) results in 20 copies of IC being delivered to each test sample.Poisson statistical analysis to determine IC copy number.
The concentrations of the IC stock solutions were quantitated by
serially diluting the stocks and performing multiple amplifications (28). At a particular dilution, the set of replicate
amplifications generated a mixture of positive and negative results. At
this low concentration, IC molecules are distributed among replicate amplifications according to Poisson's law, with some replicates having
one or a few IC molecules and others having none. The average number of
molecules in a given volume of solution (C) and the probability that no molecule exists in a particular sample of the given
volume (Pn) are given by the formula
C = 
ln(Pn) where Pn was determined by counting the number of
negative replicates and used to calculate C, the number of
IC molecules in the volume of solution added to each replicate.
[C is actually the number of signal-generating units (SGU),
which is defined as the smallest unit in the given solution that will
generate a PCR-positive signal. The physical meaning of a SGU is a
particle consisting of one or more PCR-amplifiable molecule(s). Under
ideal conditions, a SGU is equal to a single copy of the target
molecule.] The IC concentration was calculated by multiplying
C by the appropriate dilution factor.
Specimen collection and processing. (i) C. trachomatis and N. gonorrhoeae. Endocervical and male urethral swabs were collected by standard procedures and inoculated into chlamydial culture transport medium (CTM; either 2SP or MicroTest M4; MicroTest, Inc., Snellville, Ga.). A second swab was collected from each patient and cultured for N. gonorrhoeae. Specimens in CTM were transported to the lab at 2 to 8°C where an aliquot was used for Chlamydia culture. A second aliquot was shipped on wet ice to Roche Molecular Systems, where it was processed and tested on the COBAS AMPLICOR system. A 100-µl sample of specimen was mixed with 100 µl of C. trachomatis-N. gonorrhoeae lysis buffer and incubated for 10 min at room temperature. The resulting mixture was combined with 200 µl of C. trachomatis-N. gonorrhoeae specimen diluent and incubated for an additional 10 min at room temperature.
Ten to 50 ml of first-catch urine was also collected from each subject. The urine was shipped on wet ice to Roche Molecular Systems, where it was processed and tested on the COBAS AMPLICOR system. A 500-µl sample of urine was combined with 500 µl of C. trachomatis-N. gonorrhoeae urine wash buffer and incubated at 37°C for 15 min. The mixture was then centrifuged at 12,500 × g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 250 µl of C. trachomatis-N. gonorrhoeae lysis buffer. After a 15-min incubation at room temperature, 250 µl of C. trachomatis-N. gonorrhoeae specimen diluent was added to the lysate. The specimens were centrifuged at 12,500 × g for 10 min, and the resulting supernatant was tested.(ii) M. tuberculosis. Sputum specimens were collected, liquified, decontaminated, and concentrated by standard procedures. Liquified, decontaminated, and concentrated specimens were shipped to the laboratory at 2 to 25°C, where aliquots were tested for the presence of mycobacteria by culture and by acid-fast smear. An additional aliquot was processed and tested for M. tuberculosis on the COBAS AMPLICOR system. A 100-µl sample of liquified, decontaminated, and concentrated sputum was mixed with 500 µl of respiratory specimen wash solution and centrifuged at 12,500 × g for 10 min. The supernatant was discarded, and 100 µl of respiratory specimen lysis reagent was added to the resulting pellet. The resuspended specimen was incubated at 60°C for 45 min and then combined with 100 µl of respiratory specimen neutralization reagent.
(iii) HCV. Blood samples were collected in VACUTAINER serum separator tubes or in VACUTAINER blood collection tubes containing either ACD or EDTA as an anticoagulant. Collection tubes were stored at room temperature for up to 6 h until serum or plasma was prepared. Plasma and serum samples were shipped to the laboratory at 2 to 8°C, where aliquots were processed and tested for HCV on the COBAS AMPLICOR system. A 100-µl sample of serum or plasma was combined with 400 µl of HCV lysis reagent and incubated at 60°C for 10 min. The lysate was combined with 500 µl of isopropyl alcohol, incubated at room temperature for 2 min, and centrifuged at 14,000 × g for 15 min. The supernatant was discarded, and the resulting pellet was washed with 1 ml of 70% ethanol. The washed pellet was resuspended in 1 ml of HCV specimen diluent containing HCV IC. The serological status of patients was determined by enzyme immunoassay (EIA).
Amplification and detection. A 50-µl sample of processed specimen was added to 50 µl of master mix containing IC and amplified by using the thermal cycler onboard the COBAS AMPLICOR system. Each test used a unique set of thermal cycling conditions, which were automatically performed by COBAS AMPLICOR (8, 12). Upon completing amplification, the COBAS AMPLICOR system automatically denatured the amplification reaction mixtures, hybridized the amplicon to target-specific oligonucleotides bound to magnetic microparticles, and colorimetrically detected the captured amplicon by using an avidin-horseradish peroxidase complex (8, 12). The specific target and the IC were detected in separate reactions using separate, target- and IC-specific, oligonucleotide capture probes.
Where indicated, certain samples being tested for C. trachomatis and N. gonorrhoeae were amplified on a GeneAmp PCR System 9600 thermal cycler (Perkin-Elmer Corporation, Norwalk, Conn.) using the following thermal cycling parameters: 2 min at 50°C, 5 min at 95°C, and then 35 cycles, with 1 cycle consisting of 10 s at 91°C, 50 s at 62°C, and 35 s at 72°C. Amplification products were detected colorimetrically after hybridization to microwell plates coated with oligonucleotide probes specific for C. trachomatis, N. gonorrhoeae, or IC (15).Interpretation of results. Specimens yielding target signals above the test cutoff were interpreted as positive, regardless of the IC result. Specimens yielding target signals below the test cutoff were interpreted as negative, provided that the IC signal was above the assigned cutoff. Specimens yielding below cutoff signals for both the target and IC were interpreted as inhibitory. Inhibitory specimens were retested by processing another aliquot of the original specimen. The repeat test results were classified with the above criteria.
For HCV, sensitivity was calculated by comparing PCR results to EIA results. For the C. trachomatis, N. gonorrhoeae, and M. tuberculosis tests, sensitivity was calculated by comparing PCR results to resolved results. Because culture is not 100% sensitive, these tests yielded specimens that were positive by PCR but negative by culture. For C. trachomatis and N. gonorrhoeae, these discrepant results were resolved by performing PCR for an alternative target DNA sequence: a portion of the major outer membrane protein gene was used for C. trachomatis (9), and a portion of the 16S rRNA gene was used for N. gonorrhoeae (16). Specimens were considered positive for infection if the culture was positive or if the specimen was PCR positive for both the primary and alternative targets. For M. tuberculosis, discrepancies between PCR and culture results were resolved based on patient chart review; specimens were considered positive if there was clinical evidence of M. tuberculosis infection.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of primary target on IC signal. A single set of primer oligonucleotides is used to amplify both the IC and target DNA. Both targets also draw from a common pool of nucleotides and polymerase. Thus, IC amplification may be suppressed due to competition in samples containing large amounts of target DNA. We determined the amount of target DNA required to suppress the IC signal by testing a set of samples that contained increasing amounts of C. trachomatis target DNA. These samples also contained a constant low concentration (25 copies per test sample) of N. gonorrhoeae target DNA to determine whether it was similarly sensitive to competition.
The IC signal progressively decreased as the amount of C. trachomatis target DNA increased (Fig. 1). Between 250 and 2,500 copies per test sample (corresponding to 5,000 and 50,000 copies per ml of specimen) of C. trachomatis DNA was required to produce a noticeable decrease in the IC signal. The IC signal was reduced to background by 2.5 × 105 copies per test sample (5 × 106 copies per ml of specimen) of C. trachomatis target DNA. Virtually identical IC signals were observed when the N. gonorrhoeae target DNA concentration was varied and the C. trachomatis target concentration was maintained at 25 copies per test sample (data not shown).
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Determination of IC cutoff. The purpose of the IC is to maximize test sensitivity by identifying inhibitory (i.e., nonamplifiable) specimens that have the potential to generate false-negative results. The IC signal that identifies all such inhibitory specimens is chosen as the cutoff. As a first step in determining the IC cutoff, we analyzed the distribution of IC signals in a set of specimens that were negative for the target DNA. Specimens that were positive for target DNA (both true and false positives) were not included in this analysis because the depressed IC signals in these specimens did not necessarily serve as an indicator of inhibition.
Of 1,915 urogenital specimens tested by the COBAS AMPLICOR C. trachomatis test, 1,688 gave negative signals for C. trachomatis. The distribution of IC signals in these 1,688 specimens was clearly bimodal, with most specimens having an IC signal of
1.0 A660 and a smaller
set of specimens having an IC signal of <0.2
A660 (Fig. 2). IC
signals between 0.2 and 0.5 A660 were observed
in 0.8% (13 of 1,688) of the specimens; IC signals between 0.5 and 1.0 A660 were observed in 1.0% (17 of 1,688) of the
specimens. When amplification was performed in the absence of clinical
material, the IC signal was always 2.0 A660
(data not shown). These data suggest that specimens having IC
signals below 0.2 A660 are inhibitory. Specimens
having IC signals between 0.2 and 1.0 A660
probably contained weak inhibitors that partially suppressed
amplification. To select the optimal IC cutoff, we determined whether
test sensitivity was enhanced by classifying such partially suppressed
specimens as inhibitory.
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Effect of IC on assay performance. The utility of the IC was demonstrated by testing clinical specimens obtained from patients suspected of having C. trachomatis, N. gonorrhoeae, M. tuberculosis, or HCV infection. Specimens were tested as described and classified as positive or negative for infection (see Materials and Methods). Any specimen that was inhibitory (i.e., negative for both IC and target DNA) was retested by processing and testing another aliquot of the original specimen.
The fraction of specimens that were inhibitory in the test ranged from 0 to 11.5% (Table 1). Inhibition was somewhat more frequent in uninfected specimens than in infected ones. This is expected because even minimal inhibition will suppress amplification below the threshold required to generate a positive IC result. In contrast, infected specimens that suppress amplification to a similar extent will not appear inhibitory, provided that the target DNA concentration is high enough.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The results of this study demonstrate that incorporating an IC into PCR-based tests increases sensitivity by enabling the user to identify and retest samples inhibitory to PCR. Furthermore, a positive IC result indicates that amplification has occurred and thus provides assurance that negative test results are truly negative. The COBAS AMPLICOR tests evaluated in this study exhibited low rates of inhibition. Because inhibition was infrequent, use of the IC resulted in only a relatively modest improvement in test sensitivity.
The IC can also be used to monitor competition between multiple targets in multiplex PCR tests. A negative IC result in specimens that are positive for one target indicates that competition and/or inhibition reduced amplification efficiency below the threshold required to generate a positive result from a low-level target. In such specimens, negative results for the other targets are considered invalid. The data presented here demonstrate that competition will not cause a false-negative result unless the concentration of one target is 104-fold greater than the concentration of the second target.
The IC is used at a concentration of 20 copies per test sample to monitor amplification at the limit of test sensitivity. PCR inhibitors decrease amplification efficiency, thereby reducing the amount of PCR product generated from each target molecule. A high target load can compensate for reduced amplification efficiency, yielding enough product to generate a positive signal. If used at a higher concentration, the IC might not detect weak inhibition that could cause false-negative results at extremely low target loads.
For routine clinical applications, the laboratory can maximize test sensitivity by using an IC to monitor amplification in every specimen. Nevertheless, it may be possible to use an IC selectively to increase efficiency without sacrificing performance. For example, the fully automated COBAS AMPLICOR system can be programmed to perform additional tests based on the outcome of an initial test. Thus, a COBAS AMPLICOR user can easily detect the IC only in specimens that test negative for the primary target(s). Using this algorithm will not compromise test performance because the IC result has no effect on the interpretation of positive specimens.
An IC can also be used selectively when past experience has demonstrated very low rates of inhibition. Use of an IC can be reserved for specific specimen or patient conditions where inhibition is more likely to occur; examples include specimens contaminated with blood or other interfering substances and urogenital specimens from pregnant women (11). An IC should also be selectively employed when the probability of infection is high and the consequence of a false-negative result is severe. An example is testing acid-fast bacillus smear-positive patients for M. tuberculosis, where a false-negative test result would lead the clinician to conclude that the patient has a nontuberculosis mycobacterial infection.
Even when the IC was used, test sensitivity was not 100%. Thus, factors other than inhibition must have caused false-negative results. Sample-to-sample variation is one source of false-negative results. Localization of infection may also be a factor when testing female urine samples for C. trachomatis and N. gonorrhoeae because PCR and culture are performed on specimens collected from different anatomical sites. Low target concentration represents a second potential source of false-negative results, especially when testing for M. tuberculosis where the volume of sample used for PCR testing is 5% of that used for culture. Finally, the reference test result could actually be false positive. This is almost certainly a factor in HCV testing where a positive EIA result for anti-HCV antibody indicates prior exposure but does not serve as a marker for current infection.
Inhibition has been shown to affect the sensitivity of LCR-based amplification tests (2, 4-6, 11, 22, 25), TMA tests (17, 21), and a nucleic acid sequence-based amplification test (18). It is, however, difficult to determine the frequency of inhibition because the commercially available LCR- and TMA-based tests lack an IC. Consequently, inhibition can be evaluated only in reference test-positive specimens that are expected to give positive amplification test results. Inhibition cannot be assessed in reference test-negative specimens since they are expected to give negative test results. The frequency of inhibitory, positive specimens can be estimated from the frequency reference test-positive, amplification test-negative specimens. This provides an upper limit on the inhibition rate in reference test-positive specimens; the actual inhibition rate may be lower because false-negative results may be caused by factors other than inhibition. The frequency of LCR false-negative results in urogenital specimens were 2% (7 of 345) (6), 7% (3 of 44) (22), 12% (10 of 87) (5), 13% (7 of 54) (25), 22% (2 of 9) (2), and 60% (15 of 25) (11). In some instances, specimens were retested at full strength and after being diluted. Approximately one-half of the retested false-negative specimens became positive (some required dilution, while others did not); thus, the false-negative results in these specimens can be attributed to inhibition. These observations suggest that the actual LCR inhibition rate ranged from 1 to 30%. The frequency of TMA false-negative results was 10% (6 of 60) (21); 5 of the 6 false-negative specimens were positive when retested, which suggests the inhibition rate in positive specimens was 8%. For both LCR and TMA, the inhibition rate for all specimens is probably higher; weak inhibition may go undetected in positive specimens that contain relatively high concentrations of target.
In addition to monitoring amplification in qualitative PCR tests, an IC can be used as an internal quantitation standard (QS) for quantitative PCR tests (19, 27). In quantitative PCR tests, the QS is added to each clinical specimen at a known copy number and is carried through all test processes. The QS controls for specimen-to-specimen differences in recovery during processing, amplification efficiency, and detection efficiency to permit the accurate quantitation of target nucleic acid in each specimen. The amount of target nucleic acid in a specimen is calculated by multiplying the target-specific signal by the ratio of QS input copy number to QS signal.
In summary, we have developed an IC that is introduced into each test sample and is coamplified with target nucleic acid from the clinical specimen. We have used the IC to demonstrate that the frequency of inhibition in COBAS AMPLICOR PCR tests ranged from 1 to 10%. Although the frequency of inhibition is low, an IC has been incorporated into COBAS AMPLICOR and AMPLICOR tests to assure the integrity of negative results. Furthermore, by using the IC, we identified additional infections that would otherwise have gone undetected.
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ACKNOWLEDGMENTS |
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We thank E. Dragon, T. White, and R. Pinnola for helpful comments during preparation of this manuscript. J. Q. Yang and A. Wang provided clinical data for the COBAS AMPLICOR C. trachomatis and N. gonorrhoeae tests and IC cutoff data. Ann Butcher, K. Gutekunst, C. Young, V. Tevere, R. Sun, G. Colucci, M. Pedrocchi, H.-J. Burkardt, J. Vincelette, M. Bogard, J. Schirm, A. Bouza, R. Alonso, R. Antinozzi, and T. Fenner provided clinical data for the COBAS AMPLICOR M. tuberculosis and HCV tests.
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FOOTNOTES |
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* Corresponding author. Mailing address: Roche Molecular Systems, 1080 Route 202, Somerville, NJ 08876. Phone: (908) 253-7463. Fax: (908) 253-7665. E-mail: Maurice.Rosenstraus{at}roche.com.
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Abstract
Introduction
Materials & Methods
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Discussion
References
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Journal of Clinical Microbiology, January 1998, p. 191-197, Vol. 36, No. 1
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