Procedural controls for M-PCR are similar to controls that should be used for conventional PCR. These consist of both positive and negative controls, which are designed to monitor the sensitivity and specificity of each amplification run, respectively. The preparation of a batch of clinical specimens to be tested by PCR should include up to six negative specimens, depending on the number of specimens being tested. Use two negative controls for a batch of six specimens and six for a batch of 20 specimens. Possible negative controls include extraction buffer alone or negative cells or tissues interspersed with the clinical specimens to assess specimen contamination at the set-up stage. We also include one or two positive controls to ensure that the specimen preparation step worked, especially if extraction of the nucleic acid from tissue with enzymes, detergents, or organic compounds is involved. Each PCR run should include a small sensitivity panel consisting of serial 10-fold dilutions of DNA (ranging from 1 pg to 0.1 fg) to assess day-to-day fluctuation in assay sensitivity. This test is also useful in comparing results obtained in different laboratories. For M-PCR, the sensitivity panel should consist of specimens with both amplification targets and specimens containing single targets as well as appropriate negative controls. When switching lots of any reagent, each new lot should be tested by substituting the new reagent (for example, Taq polymerase, PCR master mix, or dNTPs) into the existing assay and running the sensitivity panel alone or running the sensitivity panel with a batch of specimens, and comparing the sensitivity obtained with the new reagent to that of the existing assay. Only when each new reagent is tested in this fashion and each is shown not to affect the performance of the assay adversely should any reagents in the assay be changed. This precaution is especially important when switching to a new lot of enzyme or trying out a new set of primers (perhaps purchased from a new company). If this control procedure is followed religiously, there should be few surprises necessitating costly repeat testing of large runs.
The use of good quality reagents, as well as good laboratory practices, should ensure that PCR testing generates valid results. The inclusion of quality control procedures (positive and negative controls, specimen preparation controls, sensitivity controls, monitoring for inhibitors of Taq polymerase) ensures that M-PCR results are interpreted correctly and minimizes the limitations on this technology. Although most M-PCR assays should give clearly distinguishable positive and negative results (especially when DNA probing is used to verify the presence of specific amplicons), some types of specimens can present problems. Specimens containing very high concentrations of DNA sometimes give spurious nonspecific bands or a false negative result. These specimens must be serially diluted and retested at two or three different dilutions, retested with lower concentrations of primers, or both to decrease mispriming. The effect of widely varying concentrations of target DNA on the relative amplification efficiency of each target in M-PCR has not yet been investigated systematically. This may not turn out to be a problem in the clinical microbiology laboratory where clinical specimens usually contain 103—106 viral genome equivalents and the difference in target numbers may be small. Experimentation with various amounts of each primer pair may help answer this question. Advances in specimen preparation protocols, that is, silica gel or bead adsorption of RNA for detection by RT-PCR, and the implementation of new enzymes such as Thermus thermophilus polymerase, which has both reverse transcriptase and DNA polymerase activity, have had major effects in decreasing the variability of results obtained by RT-PCR and have allowed more laboratories to detect RNA viruses reliably. Commercial tests for several RNA viruses are currently in clinical trials and will soon be available. Future technological advances will permit the detection of both RNA and DNA viruses in the same specimen by M-PCR.
If proper anticontamination measures are followed so PCR false positives are either prevented altogether or readily identified, there is little difficulty interpreting positive test results. Only when both positive and negative controls give expected results can the results of a run be accepted. M-PCR results should show clearly distinguishable bands of the predicted amplicon size by gel electrophoresis. In a well-designed and characterized M-PCR assay, gel electrophoresis may be sufficient to identify specimens as positive. Confirmatory DNA probing (either by dot blot hybridization or by solidphase DNA capture with oligonucleotide probing) may be necessary when multiple nonspecific bands obscure specific amplicons. It is good practice for laboratories to confirm positives for reportable diseases and highly communicable viral infections before reporting results; PCR testing should be included in this practice. We have used a confirmatory PCR for our in-house PCR for C. trachomatis, targeting a second plasmid sequence or a chromosomal sequence to confirm our initial PCR results (Mahony et al., 1992). The first commercially available amplification test for C. trachomatis, Amplicor™ Chlamydia from Roche Molecular Systems (Branchburg, NJ) has, however, not included a confirmatory test. Since no test is 100% specific, a small number of false positives will be reported unless a confirmatory test is employed. The presence of Taq polymerase inhibitors in clinical specimens, which results in false negative results for truly positive specimens, is worrisome but by most accounts, this is an infrequent occurrence. We and others have observed chlamydial PCR inhibitors in urine occurring at a frequency below 0.3% (Mahony et al., 1992; S. Herman, personal communication). Blood or serum specimens containing heme and semen specimens containing the polyamines spermine and spermidine (Ahokas and Erkkila, 1993) may also produce false negative PCR results because of the presence of these interfering compounds. A number of internal PCR controls including /3-globin, HLA DQa, actin, and myosin genes have been used by some laboratories to monitor the presence of inhibitors (Coultee et al., 1991; Holodniy etal., 1991; Jullian etal., 1993; Richards«?/ ai, 1993). Although this procedure doubles the number of reactions, it is an effective means of detecting inhibitors in clinical specimens that may yield false negative results.
We gratefully acknowledge the excellent contributions of Kathy Luinstra in performing PCR experiments and Lisa Rizzo and Lucia Weatherley in typing the manuscript.
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PCR in Situ Hybridization
Gerard J. Nuovo
Department of Pathology
State University of New York at Stoney Brook
Stony Brook, New York 11794
A. Review of the Methodology
B. Strengths and Weaknesses of PCR in Situ Hybridization
A. The Equivocal Penile or Vulvar Biopsy
B. In Situ Detection of PCR-Amplified HIV-1 Nucleic Acids and Tumor Necrosis Factor cDNA
C. Localization of PCR-Amplified Hepatitis C cDNA References
Haase et al. (1990) were the first investigators to describe the in situ detection of polymerase chain reaction (PCR)-amplified DNA in intact cells. This landmark work used cell suspensions in tubes. Clearly, it would be advantageous to apply this analysis to tissue sections on glass slides. This method was first reported by Nuovo et al. (1991a), who demonstrated the distribution of PCR-amplified human papillomavirus (HPV) DNA in cervical squamous intraepithelial lesions (SILs). Since this time, many groups have published protocols for PCR in situ hybridization, primarily for the detection of human immunodeficiency virus type 1 (HIV-1) DNA^Bagasra et al., 1992; Chiu et al., 1992; Nuovo, 1992; Nuovo et al., 1992a; Embretson et al., 1993a,b;
MOLECULAR METHODS FOR VIRUS DETECTION
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
Patterson et al., 1993). In 1992, Nuovo et al. reported the detection of a variety of human mRNAs in cultured cells using reverse transcriptase (RT) in situ PCR and later, in tissue sections (Nuovo et al., 1992b,d; 1993a,b). Several other groups have described the detection of mRNAs and HIV-1 RNA by RT in situ PCR; similarly, PCR-amplified hepatitis C virus (HCV) RNA (cDNA) has been detected in tissue sections (Nuovo et al., 1993a; Patterson et al., 1993).
Perhaps the greatest impact of PCR in situ hybridization is in the area of HIV-1-related pathogenesis. One of the most perplexing issues regarding acquired immunodeficiency syndrome (AIDS) is that early studies stated that few of the T helper cells actually contain the virus, especially early in the disease process. This fact is important because the reported detection rate of <1 per 1000 CD4 cells is not consistent with the severe immunosuppression that is characteristic of AIDS (Harper et al., 1986; Shapshak et al., 1990). Using different variations of the PCR in situ hybridization technique, Nuovo et al. (1992c), Bagasra et al. (1992), and Patterson et al. (1993) independently demonstrated that up to 10% of circulating peripheral blood CD4 cells are actually infected by the virus early in the disease process, and over 80% are infected in advanced AIDS. Using PCR in situ hybridization, Nuovo et al. (unpublished data) and Embreston et al. (1993a,b) showed that in early HIV infection up to 25% of the dendritic cells and over 50% of the CD4 lymphocytes in the lymph nodes contain HIV-1 DNA. At end-stage AIDS, however, virtually all CD4 lymphocytes in the lymph nodes and in the peripheral blood are infected by the virus. Clearly, these numbers are consistent with the profound immunosuppression that is the central feature in patients with AIDS.
A great deal of variation is seen in the different published protocols for PCR in situ hybridization. Much of this variation is based on methods that attempt to inhibit diffusion of the amplified product from the site of origin and to prevent drying of the amplifying solution. The use of multiple primer pairs that produce DNA segments with 40-60 bp overhangs was offered as a way to limit diffusion of the amplified product (Haase et al., 1990; Chiu et al., 1992). Although multiple primer pairs were able to increase the intensity of the signal in PCR in situ hybridization, a single primer pair could function as well if not better when the hot-start modification was used (Nuovo et al., 1991b). This finding suggested that the degree of target-specific amplification, not the size of the product, determined the success of PCR in situ hybridization. Similar conclusions about solution-phase PCR (i.e., that inhibition of the nonspecific pathways of DNA synthesis could enhance the detection of the target) were made subsequently (Nuovo et al., 1991b; Chou et al., 1992; see Erlich et al., 1991, for a review of hot-start PCR).
Much attention has focused on the ability to label the product directly in in situ PCR, which would eliminate the need for a hybridization step.
Clearly, assurance is required that incorporation of the reporter molecule is completely target specific. As will be further explained later, it appears that such assurance is possible for RNA detection in tissue sections with RT in situ PCR but not for in situ PCR methods for DNA in tissue sections (Nuovo, 1992,1993).
B. Strengths and Weaknesses of PCR in Situ Hybridization
PCR in situ hybridization has three major strengths. First and foremost, PCR in situ hybridization combines the high sensitivity of PCR with the cell localizing ability of in situ hybridization. Why does one need to amplify the product to detect it by in situ hybridization? Researchers generally agreed that in situ hybridization is less sensitive than filter hybridization techniques and PCR (Lorincz et al., 1989; Nuovo, 1989,1992,1993). Many studies have compared the sensitivities of these three techniques, often with respect to the detection of viruses associated with infectious diseases, particularly HPV and HIV-1. These studies have strongly suggested that (1) PCR, filter hybridization, and in situ hybridization are of equivalent sensitivity for productive viral infections, such as HPV in low-grade SILs; and (2) latent infection, as routinely noted in HIV-1 infection, and occult or subclinical infection can be detected by PCR or filter hybridization but not by in situ hybridization (Lorincz et al., 1989; Nuovo, 1989,1992,1993). This result reflects the lower copy number of virus associated with these specific conditions. The reported detection thresholds of the three techniques vary considerably, in part reflecting methodological variations. For example, the hot-start modification of PCR increases the detection threshold from 1000-2000 copies to 1-10 copies, assuming 1 fig background nontarget DNA (Nuovo et al., 1991b; Chou et al., 1992). The detection threshold for filter hybridization is generally reported to be about 1 copy per 100 cells (Lorincz, 1989; Nuovo, 1989,1992,1993). Most groups report detection thresholds for in situ hybridization of about 10 copies per cell (Crum et al., 1988,1991; Walboomers et al., 1988; Nuovo et al., 1991a). Claims that 1 copy per cell can be detected by in situ hybridization have been made (Lawrence et al., 1990). In our experience, such reports are in conflict with the general inability to detect latent HIV-1 infection by in situ hybridization, which is associated with 1 to a few integrated copies of DNA (Bagasra et al., 1992; Nuovo et al., 1992c,1993b; Embretson et al., 1993a,b; Patterson et al., 1993). Interest in PCR in situ hybridization has grown out of the experience of virtually all investigators that the relatively high detection threshold of in situ hybridization is a major limiting factor for its usefulness.
A second strength of PCR in situ hybridization relates to the issue of sample contamination in solution-phase PCR. Sample contamination, which can lead to false positive results in PCR, has limited its usefulness as a diagnostic test. However, this problem has not been encountered in PCR in situ hybridization because the positive signal is localized to specific areas within the cell. Indeed, we have added purified viral DNA to cell and tissue specimens known to be negative for the virus and shown that the PCR product remains in the amplifying solution and does not enter the fixed cell.
The third strength of PCR in situ hybridization is the enormous amount of information it provides relative to the histological distribution of the amplified PCR product. For example, the observation that PCR-amplified HIV-1 DNA was restricted to the endocervical aspect of the transformation zone in the cervix suggested that this area may be its portal of entry (Nuovo et al., 1993). Similarly, the demonstration that HPV RNA, as evident from RT in situ PCR, was found in most cancer cells but in none of the adjacent normal cells suggests that viral transcription is essential for the malignant phenotype (J. Chumas and G. J. Nuovo, unpublished observations).
The weakness of PCR in situ hybridization is related to the weaknesses of both PCR and in situ hybridization. Competing pathways in PCR can limit its specificity and sensitivity. Further, background signal from complexing of the probe and nontarget molecules in in situ hybridization can also lead to false positive results (Nuovo, 1992,1993). The need for controls for every slide will be stressed in this manuscript as a way to ensure that the conditions are optimized for PCR and that background is not causing problems in interpreting the hybridization signal.
As noted earlier, methods for the in situ detection of PCR-amplified DNA have been reported by several groups. This chapter describes a manual "hot-start" modification for PCR in situ hybridization that allows the detection of one target copy per cell with a single primer pair (Nuovo et al., 1991b, 1992a,e; Nuovo, 1992). This modification reduces much of the unwanted DNA synthesis due to mispriming and primer oligomerization and, under certain specified conditions, permits direct incorporation of target-specific labeled nucleotides in a process termed in situ PCR. This process has been expanded to use for RNA detection by preceding PCR with an RT step.
The protocols listed here for the in situ detection of PCR-amplified DNA and cDNA are derived from two textbooks (Nuovo, 1992,1993).
1. Preparation of Tissue Sections and Cell Samples
1. Fix tissue samples in 10% buffered formalin, preferably 8-15 hr.
2. For cell cultures, wash the cells directly in the plates. Add 10% buffered formalin and let stand overnight. Place 2000-5000 cells per 1-cm area using a cytospin centrifuge.
3. Place several 4-^u.m tissue sections or 2 cytospins on silane-coated slides (Oncor, Gaithersburg, MD)
4. To remove the paraffin from tissue samples, place slides in fresh xylene for 5 min and then in 100% ethanol for 5 min. Then air dry.
Pepsin, trypsin, or proteinase K may be used. Proper protease concentration and time needed for digestion are based on tissue type and length of fixation and are established by trial and error. Insufficient protease treatment can result in lack of hybridization signal, whereas excessive protease treatment will cause destruction of tissue morphology.
1. Begin with pepsin at 2 mg/ml (stock solution is 20 mg pepsin, 9.5 ml water, 0.5 ml 2 TV HC1). Treat the cells or tissue for 12-30 min.
2. For RT in situ PCR, inactivate using sterile diethylpyrocarbonate (DEPC)-treated water for 1 min followed by 100% ethanol for 1 min.
3. For PCR in situ hybridization, inactivate using a solution of Tris (0.1 M pH 7.5) and 0.1 M NaCl for 1 min followed by 100% ethanol for 1 min.
1. Treat the RNase-free DNase (Boehringer Mannheim, Indianapolis, IN) overnight. Use 10 U/tissue section. Cover the solution with a polypropylene cover slip and incubate at 37°C.
2. Inactivate the DNase by washing the slides in DEPC-treated water for 1 min, followed by 100% ethanol for 1 min. Then air dry.
2 ¡A each of dATP, dCTP, dGTP, and dTTP (stock solution: 10 m M) 1 fil of 3' downstream primer (stock solution: 20 fiM)
3 fil DEPC-treated water 1 fil RNase inhibitor
4 fil MgCl2 (stock solution: 25 m M) 1 Ail (2.5 U) RT
This is enough for 2 tissue sections. (Note: This solution is prepared as described in the RT PCR GeneAmp kit; Perkin-Elmer Corporation, Nor-walk, CT.)
4. Add 10 fil to each tissue section and incubate at 42°C for 30 min.
1. For amplifying solution add to one tube:
2.5 /A PCR Buffer II (GeneAmp kit) 4.5 fil MgCl2 (stock solution: 25 mM)
4 fil dNTPs (stock solution: 2.5 mM; dilution as per GeneAmp kit) 1 fil primer 1 (5' primer; stock solution: 20 fiM) 1 fil primer 2 (3' primer; stock solution: 20 fiM) 1 fil bovine serum albumin (BSA; stock solution: 2% w/v) 6.2 ¡A water
2. To a separate tube, add 4.0 water and 0.8 /xl Taq polymerase (AmpliTaq, Perkin Elmer).
3. Cover each of two tissue sections with 10 fil amplifying solution. Cut plastic cover slips to size and cover the amplifying solution. Then anchor the cover slips with 2 small drops of nail polish.
4. Place slides in an aluminum foil boat, and then on the block of an automated thermal cycler (Perkin Elmer).
5. When the block has reached 55°C, add 2.4 ¡il Taq polymerase solution to each section by gently lifting the cover slip. Immediately overlay the section with about 1 ml preheated (82°C) mineral oil.
6. Bring block to 82°C, then to 94°C for 3 min (denaturing).
7. Incubate at 55°C, 2 min and 94°C, 1 min for 30 cycles; add more heated mineral oil as needed.
9. Remove cover slip and oil by soaking in xylene for 5 min followed by 100% ethanol for 5 min. Then air dry slides.
5. In Situ Hybridization
1. Label oligoprobe using the Genius 5 oligoprobe 3' tailing kit (Boeh-ringer Mannheim, Indianapolis, IN).
2. Make oligoprobe cocktail by adding 10 (A formamide, 39 ¡A 25% dextran sulfate, 39 ¡A water, 10 ¡A 20x SSC, and 2 ¡A probe.
3. Add 5-10 ¿c.1 probe cocktail to a given tissue section.
4. Overlay with plastic cover slip.
6. Place slides in humidity chamber at 37°C for 2 hr.
7. Remove cover slip; place slide in wash solution (lx SSC and 0.2% BSA) for 10 min at 50°C.
8. Wipe off excess wash solution.
9. Add 50 fA anti-digoxigenin-alkaline phosphatase conjugate per tissue section.
10. Incubate in humidity chamber for 30 min at 37°C.
11. Wash slides at room temperature for 3 min in the detection solution (0.1 M Tris, pH 9.5, 0.1 M NaCl)
12. Place slides in detection solution and nitrobluetetrazolium/bromo-chloroindolyl phosphate (NBT/BCIP; Oncor).
13. Incubate slides for 30 min to 2 hr, checking results periodically under microscope; counterstain with nuclear fast red.
The protocols presented here reflect the results of a large series of optimizing experiments for the different reagents (Nuovo et al., 1993c). Note that the concentration of MgCl2 (4.5 mM) is uniformly higher than for solution-phase PCR. This general statement applies to a wide variety of primer pairs and DNA and cDNA targets (Nuovo, 1992; Nuovo et al., 1993c).
Diffusion of the amplified cDNA from the site of origin is another concern. Researchers have shown that the specific fixation chemistry has a profound effect on diffusion of the PCR product. To address this problem, some investigators hypothesized that the use of multiple primer pairs or tailed primers would result in a product greater than 1000 bp which would be too large to permeate the nuclear membrane (Haase et al., 1990; Chiu et al., 1992). A 450-bp product was thought to be membrane permeable. An alternative direction taken by others to solve this problem was based on the nature of cell fixation during preparation of samples, and subsequent adjustment of protease conditions. Studies examining different fixation methods for in situ PCR showed that fixatives such as acetone or ethanol, although excellent for solution-phase PCR, were unsuccessful for in situ PCR (Nuovo et al., 1993c). Acetone and ethanol are denaturing fixatives that do not crosslink nucleic acids to proteins, as do fixatives such as formalin. The non-cross-linking fixatives may not allow in situ PCR or, alternatively, amplification may occur but the PCR product diffuses from the nucleus into the amplifying solution. Analysis of the amplifying solution showed that the PCR product was indeed located in the solution when the cells were fixed in ethanol or acetone, but not when the cells were fixed in buffered formalin (Nuovo et al., 1993c). These data illustrate the need for prolonged formalin fixation and protease digestion. Formalin fixation is thought to create a migration barrier that prevents diffusion of PCR products through cell membranes. This ability is shown graphically in Fig. 1. However, this ability to prevent diffusion necessiates treatment of the samples with protease digestion to permit PCR reagent entry. The degree of inhibition of migration appears to be marked, based on studies involving cDNA from measles and a variety of human mRNAs that have showed distinct and appropriate subnuclear (i.e., nucleolar and perinucleolar) and cytoplasmic localization (Fig. 2); Nuovo et al., 1992d).
In our experience, the most common source of difficulties for PCR in situ hybridization and RT in situ PCR is protease digestion. Different tissue samples may vary greatly in their susceptibility to protease digestion and the conditions should be determined empirically. Overdigestion results in a loss of cell morphology and the nuclei are no longer visible. Inadequate protease digestion can be recognized by an absence of signal or a weak signal in the positive control. Positive controls can be prepared in two ways. First, one can use a probe that will detect a target in every human cell. Many companies sell probes that detect the repetitive Alu sequence that makes up about 5% of the total human genome; we have used these probes from Digene Diagnostics (Beltsville, MD) and Oncor with good results. This positive control tests the in situ hybridization and detection procedures.
The other type of positive control is based on the ability to incorporate digoxigenin dUTP nonspecifically into tissue sections using PCR. If the tissue is not DNase treated and protease conditions are optimal, at least 50% of the cells will show an intense signal (Fig. 3). This figure also shows the different histological distributions of the positive control (all cell types positive) and the test section, in which only the carcinoma cells in this cervical cancer are positive for HPV 18 RNA. If few of the cells show a weak signal
1. Formalin fixation creates protein - DNA links
100 base pairs
2. Amplified product remains in cell - trapped on + charged amino acids
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