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Applications Of The Polymerase Chain Reaction (PCR) Technique And Instrumentation

The Polymerase Chain Reaction (PCR) technique has had a significant impact in the field of medical genetics, it not only allows a qualitative analysis of pathogenic organisms, but also is an excellent tool to determine the viral or bacterial load of a patient.  Besides this the genotype of an organism can also be determined reliably and thus one does not have to rely on indirect methods of determining the genotypes such as serotyping.

HEPATITIS C VIRUS QUALITATIVE TEST: The Roche AMPLICOR® Hepatitis C Virus (HCV) Test version 2.0, that we routinely use in the PCR Lab. of the Shifa College of Medicine is a highly reliable qualitative in vitro assay that allows the detection of the HCV genome in patients infected with the virus.  This can be done even prior to sero-conversion and also in the case of acute HCV infection where individuals may fail to produce antibodies to HCV or in immunocompromised patients.  The test involves the isolation of the Ribonucleic Acid (RNA) genome of the virus from patient serum or plasma by lysis of the virus particle and precipitation of the RNA.  This RNA molecule is then re-suspended and is first converted to complementary Deoxyribonucleic Acid (cDNA) by reverse transcription, which is then subjected to a PCR amplification protocol with the help of specific primers, which recognize and bind only highly conserved portions of the HCV genome.  After amplification of the target the double stranded amplicon obtained is denatured and hybridized to complementary HCV oligonucleotide capture probe bound to the wells of microwell plates.  This hybridization process is then detected colorimetrically by the addition of an Avidin-Horseradish Peroxidase Conjugate which recognizes and binds to a biotin molecule bound to one of the primers used in the PCR amplification process.  This results in a higher specificity (99.3%) and sensitivity (99.5%) as compared to a regular PCR assay.  Another advantage that the Roche AMPLICOR® HCV Test has over other commercial and in-house HCV PCR kits is that in the amplification process deoxyuridine triphosphate is used instead of thymidine triphosphate this allows the selective amplification of clinical samples because any cross contaminating DNA from previous rounds of amplifications is recognized (due to the presence of deoxyuridine) and destroyed by the enzyme uracil-N-glycosylase.  This enzyme is included in all PCR amplifications and prior to the start of the amplification process destroys any contaminating amplicon but not the target clinical RNA which contains only uridine and not deoxyuridine in its sequence.  This coupled with good laboratory practices and appropriate controls, almost eliminates false positive results.

Due to a number of different factors the probability of getting a false negative result is slightly higher than that of a false positive.  The most important of these factors is the storage and transportation of the sample to the lab; whole blood must be transported at 2-25oC and processed within 6 hours of collection.  Plasma or serum may be transported at 2-8oC or frozen at -70oC or colder.  The RNA genome of HCV is quite labile and if not processed properly or in a timely fashion it degrades, thus giving a false negative result.  Another factor is that the Taq polymerase used in the PCR process is inhibited by Heme and Heparin, thus samples that are hemolyzed or those that are collected in Heparin will also give false negative results.  These and other inhibitors that may be present in clinical samples, as well as RNA loss during preparation can reduce the efficiency of the PCR process.  In order to eliminate this procedural problem so that the possibility of false negative results is further reduced, we at the Shifa College of Medicine have started using an Internal Control Detection method.  In this method a known control is added to all the samples at the time of RNA isolation, this control is then processed in parallel with the sample and detected separately.  In an inhibitory sample or one that has lost RNA during preparation this control will also give a negative result.  In these cases a fresh sample is requested from the patient that is reanalyzed free of cost to him.  We are the only PCR diagnostic laboratory in Pakistan using the Roche AMPLICOR® kit that has started using the Internal Control Detection to reduce false negative.  And although this significantly increases our cost per test, as compared to the other laboratories, this added cost has not been passed on to the patient.  We now expect our results to be more reliable by further reducing the possibility of getting false negatives.

Another factor which could result in false negatives is the primers not binding to the target and thus the absence of a PCR signal.  Although these sites in the HCV genome are located in a highly conserved region still there is always a possibility (though very small) that mutations can occur at these site and this will thus result in the primers not binding.  Thus because of an absence of primer binding there will be no amplification and therefore a false negative result will be obtained.  Obtaining a positive result is also dependent upon the number of particles in the reaction (viral load of the patient), although the Roche AMPLICOR® HCV Test is more sensitive than other PCR protocols still its lower limit of detection is 50 viral particles per ml of serum/plasma or 2.5 particles per PCR.  A sample which has a viral load lower than this will not yield a positive result.

With all the limitations mentioned above (which can be minimized or eliminated in most cases) PCR is still the method of choice to determine the presence or absence of the virus and is diagnostically more relevant as opposed to the ELISA anti-HCV antibody screening test, since PCR directly analyses the presence of the virus particle as opposed to the later test which looks for the presence or absence of the antibody response that is mounted against the organism.  Thus after exposure to HCV, PCR is the first test that will become positive and is of immediate diagnostic use whereas it will take a few weeks time before the antibody response is mounted and an individual will become antibody positive, in immune compromised patients even this will not happen.  In addition to this as a result of interferon therapy even though the virus might be cleared and the individual becomes PCR negative, he or she will still stay antibody positive as immunoglobulins have a half life of 23 days and it will be a few months time before all of the circulating antibody is cleared from the patients circulation.

 HEPATITIS C VIRUS QUANTITATIVE TEST:  In addition to the HCV qualitative test described above, Roche also markets a quantitative kit under the brand name of AMPLICOR® HCV MONITORTM, this has all the built in qualities described above for the qualitative kit.  The quantitative kit is not as sensitive as the qualitative kit; the lower limit of detection is 600 IU/ml.  In order to accurately determine the copy number/concentration of the HCV particles this kit makes use of an internal control (Quantitation Standard); known concentrations of which is introduced at the first step of RNA isolation and is then reverse transcribed, PCR amplified, hybridized and detected along with the test sample.  The obtained levels of this amplicon is then used to compensate for effects of inhibition and controls for the variability in the amplification process, this allows the accurate quantitation of HCV RNA in each clinical specimen.  The HCV quantitative test in conjunction with clinical presentation and other laboratory markers is an invaluable tool in assessing viral response to antiviral treatment as measured by changes in serum or plasma HCV RNA levels.  Data of Orito et al. (1995, Journal of Medical Virology 46: 109-115) suggests that early changes in serum/plasma HCV RNA levels may predict long-term response to interferon therapy.  As opposed to HCV RNA PCR quantitation, the measurement of alanine aminotransferase (ALT) levels is considered to be a surrogate indicator of HCV infection, but is not a direct measure of viremia.  The degree of ALT elevation cannot be directly correlated with the level of HCV infection.  In fact ALT can be elevated due to a number of causes of liver inflammation including, but not restricted to, viral hepatitis.  In contrast detection and quantitation of HCV RNA by PCR amplification offers a measure of active viremia.

For the HCV qualitative as well as the quantitative assay, blood samples should only be collected in sterile Serum Separation Tubes or in sterile tubes using ACD or EDTA as anticoagulant.  Specimen’s anticoagulated with heparin are unsuitable for these tests.  In order to ensure the accuracy of the results, along with all batches of the HCV qualitative and quantitative tests appropriate quality as well as negative and positive controls are used, in addition in the HCV quantitative tests low and high controls are also used.

 MYCOBACTERIUM TUBERCULOSIS QUALITATIVE TEST: The AMPLICOR® Mycobacterium tuberculosis test is a qualitative in vitro diagnostic test for the detection of M. tuberculosis (MTB) in clinical specimens.  The test like the HCV PCR test utilizes the PCR nucleic acid amplification and hybridization techniques for the detection of MTB in liquefied, decontaminated and concentrated human respiratory specimens, including expectorated and induced sputum, bronchial washings and bronchial alveolar lavages (BAL).  The isolation of an organism from the MTB complex is required for the definitive diagnosis of tuberculosis.  Routine cultures are time-consuming and can take up to eight weeks.  Microscopic examination of acid-fast smears is the most rapid method for the detection of mycobacteria, but it is insensitive and non-specific.  Immunological and serological techniques are limited, in general, due to poor sensitivity and/or specificity (Daniel, T.M. 1990.  Journal of Laboratory Clinical Medicine 116: 277-282 and Butler, W.R. et al. 1990.  Journal of Clinical Microbiology 28: 1204-1213).  The analysis of mycolic acids from mycobacteria by high-performance liquid chromatography is a useful adjunct to culture and biochemical testing.  Species specific nucleic acid probes have significantly improved the opportunity for rapid confirmation of culture results for several mycobacterial species.  The development of PCR-based tests specific for mycobacteria has been shown to further improve the rapid diagnosis of tuberculosis by allowing the direct detection of mycobacteria in clinical specimens (D’Amato, R.F. et al. 1995.  Journal of Clinical Microbiology 33: 1832-1834).

 The AMPLICOR® MTB test has all the controls and qualities mentioned above for the Roche AMPLICOR® Hepatitis C Virus Test.  All respiratory specimens, including expectorated and induced sputum, bronchial washings and bronchial alveolar lavages should be collected in sterile 50ml conical tubes and transported to the laboratory at 4 oC within 24 hours of collection.

 INSTRUMENTATION: There are a variety of PCR instruments available in the market to suite the needs of an advanced as well as a not so advanced user who only needs to use basic PCR amplification cycles.  Of the various companies making these instruments the most reliable ones are those of Applied Biosystems/Perkin Elmer who are the oldest manufacturers of PCR instrumentations.  The GeneAmp PCR systems 9700 and 2700 are the two most recent and reliable automated instruments of this company, of which the former is for advanced users, thus slightly more expensive and the later for the not so advanced users and also a lower price tag ideal for the cost conscious lab managers.

The 9700 has an interchangeable sample block with 60, 96 or dual-384 wells, whereas the 2700 has an integrated 96-well sample block.  Both of these instruments use an internally housed Peltier heating/cooling unit and for routine PCR amplification applications an aluminum sample block is used that allows optimal thermal transfer.  In addition the 9700 also comes with a gold-plated silver sample block that has been engineered for maximum performance and durability, utilizing a rapid heat transfer design of electroformed silver to maximize heating/cooling rates and goldplating for maximum durability.  This allows the gold-plated silver 96-well 9700 system to be used in MAX Mode (5 °C/sec average heating/cooling of block).  The 9700 as well as the 2700 have an easy-to-use, intuitive user interface as well as platinum sensors that provide a wide temperature range (4 °C to 99.9 °C), accuracy (±0.25 °C from 35 °C to 100 °C), long term stability and high reliability.  In addition the 2700 can be “packed” side-by-side on the bench because it has air vents at the back rather than the sides and a small foot prints.  The 9700 has networking and high instrument diagnostic capabilities as well as the choice of setting variable ramp rates and cycling speeds.  In addition it has a PCMCIA for software upgrade and Methods Transportability card.

 Applied Biosystems are ably represented in Pakistan by Analytical Measuring Systems who are the oldest retailers of PCR instruments in the country thus they also posses the most reliable network, backup support as well as a highly trained and reliable engineering department that easily handles all the breakdowns of the instruments; which are at an order of magnitude less than that of instruments being marketed by other companies.  We have been using the 2700 for the last more than a year with excellent results and no breakdowns at all.  In case of electricity failure although the instrument restarts from the point of the power loss, still I would recommend the use of a good quality UPS

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