Diagnostic accuracy of nucleic acid amplification tests. Pcr polymerase chain reaction is a technique in molecular genetics that permits the analysis of any short sequence of dna or rna even in samples containing only minute quantities of dna or rna. Diagnosis of the pulmonary tuberculosis by polymerase. The pcr technique can reduce the diagnosis time and may increase the detection of mycobacteria in smearnegative tb. Role of pcr method using is6110 primer in detecting. The technique is simple and may be performed in any laboratory a low sensitivity is its only limitation. Diagnosis is difficult because of unspecific clinical manifestations and low accuracy of conventional tests. This study was conducted to determine the efficiency of polymerase chain reaction pcr testing for detecting tb lymphadenitis in samples obtained by ebustbna. Nucleic acid amplification techniques such as pcr are very useful in the rapid diagnosis of infection caused by mycobacterium tuberculosis.
There is a need for rapid and sensitive detection of mycobacterium tuberculosis in tissue specimens. For this crosssectional study, 150 consecutive patients with pleural effusion. Tb are diagnostic techniques to demonstrate the presence of tubercle bacilli by the extraction and amplification of dna or rna of m. Jun 12, 2018 rt pcr reverse transcriptasepolymerase chain reaction is a highly sensitive technique for the detection and quantitation of mrna messenger rna. Tuberculosis tb, whooping cough, brain abscesses and spinal infection, otitis media with effusion, mycoplasmal pneumonia, endophthalmitis and bacterial meningitis.
Polymerase chain reaction pcr is a technique used in molecular biology whereby small amounts of target dna are amplified by a chain reaction, which produces thousands or millions of copies. Rapid detection of mycobacterium tuberculosis complex by realtime. Of the 60 formalinfixed, paraffinembedded tissue specimens studied. Naat for tb diagnosis q becoming standard of practice qcannot replace clinical judgment or be relied on as the only guide for therapy or isolation practices less than perfect sensitivity and specificity q sensitivity q95% for afb smear positive tb patients q5575% of afb smear negative, culture positive tb patients.
Tb laboratory techniques for diagnosing tuberculosis dale e. While the xpert mtbrif assay cepheid sunnyvale, ca, usa has greater than 95% sensitivity for identifying acidfast bacilli afb smearpositive tuberculosis tb cases, for smearnegative pulmonary tuberculosis ptb the sensitivity was as low as 60% 1, 2. The full text of this article is available as a pdf 191k. Quantitative pcr qpcr is a modified version of this technique where the amount of dna can be quantified during the. Jul 23, 2014 laboratories can diagnose tb in a number of ways.
Intended use quantiferon tb gold qft is an in vitro diagnostic test using a peptide cocktail simulating esat6, cfp10, and tb7. The polymerase chain reaction pcr is a laboratory technique for dna replication that allows a target dna sequence to be selectively amplified. Novel technique of quantitative nested realtime pcr assay. The current standard is a positive mycobacterial culture from a body fluid. Pdf the present study was carried out for the detection and identification of bovine tuberculosis btb using polymerase chain reaction pcr. Conversely, fully calcified, discrete, nodular lesions without fibrosis. Transmission of the disease to humans is through direct contact with diseased animals and consumption of unpasteurized milk and milk products. Pleural tuberculosis tb diagnosis often requires invasive procedures such as pleural biopsy. Laboratory tests can either support or confirm clinical symptoms of tb. Thus, it is a quicker, more sensitive technique, which also presents a lower risk of causing environmental contamination6,8. The diagnosis of tuberculous meningitis tbm remains a complex issue because the most widely used conventional diagnostic tools, such as culture and pcr assay for cerebrospinal fluid csf samples, are unable to rapidly detect mycoba ct erium tuberculosis with sufficient sensitivity in the acute phase of tbm. The polymerase chain reaction pcr is a method of replicating dna, it makes numerous copies of a specific segment of dna quickly and accurately 1. It requires prior knowledge of a dna sequence, including differences between alleles, and uses primers whose 3 ends encompass the snp. The aim of this study was to compare the polymerase chain reaction pcr and culture methods for the detection of m.
However, variations in procedures for inhouse pcr could explain the wide variability of sensitivity and specificity reported in several studies. These tests vary in which nucleic acid sequence they detect and vary in their accuracy. Using pcr, copies of dna sequences are exponentially amplified to generate thousands to millions of more copies of that particular dna segment. Pdf urinary polymerase chain reaction for diagnosis of. Pcr technique with its application open access journals.
Polymerase chain reaction, diagnosis, bacteria, infections introduction the polymerase chain reaction pcr is a technique widely used in molecular biology. Application of eric pcr for the comparison of isolates of. The pcr data were compared with conventional microscopy and culture technique and also with results from existing tbpcr kit. One hundred and twenty sputum samples were randomly collected from ptb patients visiting the tb clinic in kem hospital, mumbai during 2008 to 2010 and were mostly from fresh. Diagnosis of the pulmonary tuberculosis by polymerase chain. Genebased diagnosis of tuberculosis with a newgeneration. Apr 20, 2014 pcr technique polymerase chain reaction, animation.
In this section, an overview of the principles of pcr assay techniques is. The octal designation is the form of the result that is reported by the genotyping laboratories to tb programs. About the test a commercial molecular assay to detect. Objectives qdescribe molecular tests for the diagnosis of tuberculosis and the detection of drug resistance in mycobacterium tuberculosis complex mtbc quse casebased scenarios to explain the use of molecular test results and to illustrate the benefits. A retrospective study involving a cohort of 155 patients being investigated for tuberculosis in an infectious disease consultation service was undertaken. Immunopcr ipcr is basically similar to elisa, but instead of using an enzymeconjugated antibody, the antibody is labeled with dna fragment through streptavidinbiotin conjugates or by a covalent binding, and which is amplified by pcr fig. Polymerase chain reaction, 122004 5 mgcl 2 the concentration of mgcl 2 influences the stringency of the interaction between the primers and the template dna. Before introducing any molecular technique in a diagnostic laboratory. Diagnosis of extrapulmonary tuberculosis by pcr pathogens. Pcr, applications, molecular identification, cloning created date.
Diagnostic value of mycobacterium tuberculosis dna detection by. From 2004 to 2010 total 766 different types of smear negative. Immunopcr, a new technique for the serodiagnosis of. An additional advantage of realtime pcr is the relative. Doctors give unbiased, trusted information on the benefits and side effects of screen to treat mycobacterium tuberculosis infection. The method was applied to blood samples from immunocompetent indi viduals with localized pulmonary tuberculosis. During the last two decades, an unprecedented interest has arisen in designing new tb diagnostic tools including nucleic acid amplification tests such as polymerase chain reaction pcr and interferongamma release assays igras, cattamanchi et al. Rtpcr reverse transcriptasepolymerase chain reaction is a highly sensitive technique for the detection and quantitation of mrna messenger rna. Application of pcr techniques for tb diagnosis at the insititute of nuclear medicine, molecular biology and oncology inmo university of gezira. It is capable of taking a small amount of dna or even single molecule and amplifying a specific region exponentially such that once the reaction is finished, there may exist up to 230 copies of. Immunopcr, a new technique for the serodiagnosis of tuberculosis.
Therefore, different types of body fluids and tissues can be used for the detection of mtb. A total of 314 samples 286 sputum and 28 bronchoalveolar lavages from 242 pa tients were evaluated by pcr, and the results were compared with the those obtained by acidfaststained. The utility of polymerase chain reaction pcr in the diagnosis of pulmonary tuberculosis. Polymerase chain reaction pcr is a method widely used in molecular biology to rapidly make millions to billions of copies of a specific dna sample, allowing scientists to take a very small sample of dna and amplify it to a large enough amount to study in detail.
Multiplex polymerase chain reaction for diagnosis of. To simplify recording, the band pattern is converted to a series of 1s and 0s 1 means that the band is present and 0 means it is absentsee the rows labeled binary code in figure 3. A polymerase chain reaction pcrbased assay for the diagnosis of tuberculosis was evaluated in 60 formalinfixed tissue specimens, the target for the amplification being a segment of is6110 in the m. Designing polymerase chain reaction pcr technique for the. Significant improvements in understanding of molecular biology of mycobacterium tuberculosis has led to development of newer diagnostic techniques for tuberculosis, and polymerase chain reaction pcr is an emerging diagnostic tool for diagnosis of tb. The synthesis of cdna complementary dna from rna by reverse transcription rt and. Designing polymerase chain reaction pcr technique for. The diagnostic techniques for the detection of mycobacterium include polymerase chain reactionpcr test. The sensitivity of pcr, culture, and staining was 97 %, 88%, and 65 %, respectively, the specificity was tuberculosis tb is presently an important h ea lth. Naats for tb are a heterogeneous group of tests that use either the polymerase chain reaction pcr technique or transcriptionmediated amplification tma or other forms of nucleic acid amplification methods to detect mycobacterial nucleic acid. The advantage of the pcr method is that tbsa in samples or tb culture can be more rapidly detected than elisa.
Pdf application of pcr techniques for tb diagnosis at the. However, its role in daytoday clinical practice is not completely clear. Diagnosing active tb disease tb testing blood test for tb. Jan 15, 2020 the polymerase chain reaction pcr is a laboratory technique for dna replication that allows a target dna sequence to be selectively amplified. Introduction quantitative reverse transcription pcr qrtpcr is a highly sensitive and versatile technique that is widely used for measuring gene expression in tissues and cultured cells. Cdc should explore validating and deploying selected analyte specific reagent asr tests for tb to public health laboratories in a manner similar to that of bioterrorismrelated asr tests. Unfortunately, the delayed diagnosis impacts the urinary tract severely. Tb disease have one or more symptoms that lead them to seek medical care.
Apr 14, 2015 with the demonstrated advantages of using this technique such as capacity to identify the m. It monitors the amplification of a targeted dna molecule during the pcr i. Use of the polymerase chain reaction for the detection of mycobacterium tuberculosis tb pcr as a basis for making clinical decisions on the initiation of antituberculosis treatment was studied. Role of molecular methods in tuberculosis diagnosis and. Use of real time polymerase chain reaction for detection. Recommendations nucleic acid amplification lab info. Application of pcr for the detection of bovine tuberculosis. Diagnosis of tuberculosis by pcrbased amplification of mpt64. Role of molecular methods in tuberculosis diagnosis and treatment. Pcr is now a common and often indispensable technique used in medical. Molecular assays based on nucleic acid amplification techniques, such as polymerase chain reaction, have been developed for rapid tb diagnosis and are being implemented in developing countries. Background endobronchial ultrasoundguided transbronchial needle aspiration ebustbna is a diagnostic method for tuberculosis tb. The aim of this study was to evaluate the role of realtime polymerase chain reaction pcr for the is 6110 sequence of m. Pcr was invented in 1983 by the american biochemist kary mullis at cetus corporation.
Pcr assay detects mycobacterium tuberculosis complex dna mtbc dna directly from pulmonary and extrapulmonary samples. Genitourinary tuberculosis is the third most common form of extrapulmonary tuberculosis. Priority should be given to rapid detection tests, such as a realtime pcr test for tb, and tests for molecular detection of rifampin resistance. A realtime polymerase chain reaction realtime pcr, also known as quantitative polymerase chain reaction qpcr, is a laboratory technique of molecular biology based on the polymerase chain reaction pcr. Blood or bone marrow collect into edta tube if unable to put directly into tb blood bottle available from laboratory on request, phone 88352. The method was applied to blood samples from immunocompetent individuals with localized pulmonary tuberculosis.
Evaluation of dna extraction techniques for detecting. Various primers are being tested for the detection of mycobacterium tuberculosis dna. Michigan department of community health tb mycology lab manager objectives overview of laboratory role in tb diagnosis and control techniques for diagnosing tb additional lab results critical to effective case management relevance of lab results to clinical and. From 2004 to 2010 total 766 different types of smear. The representative dna amplification method is the pcr assay technique. Clinical evaluation of the polymerase chain reaction for. In this study, we prospectively used the pcr technique for the detection of mycobacterium. Pdf application of pcr for the detection of bovine. Pcr is used to reproduce amplify selected sections of dna or rna for analysis. With the demonstrated advantages of using this technique such as capacity to identify the m. All smear and culturepositive samples were pcr positive.
Nucleic acid amplification tests yield fast results, and among these, new technologies can also detect drug resistance. It is a technique used to make multiple copies of a dna segment of interest, generating a large amount of copies from a small initial simple. Tuberculosis spread to almost every organ of the body and is characterized on the basis of localization of infection. The amplification of a specific cdna by the polymerase chain reaction pcr. To address the low sensitivity of current tb diagnostics for afb smearnegative cases, we developed a simple and label. Quantx onestep qrtpcr tb green kit user manual 050919 takara bio usa, inc. Recent studies, including those mentioned above, report a high accuracy of multiplex pcr for different forms of eptb like lymph node, skin, osteoarticular, and meningeal tuberculosis. Pcr may be an advance for tb peritonitis, and should help augment the adenosine deaminase test on peritoneal fluid, which is said to have 86% sensitivity and 100% specificity. In contrast to regular reverse transcriptase pcr and analysis by agarose gels, realtime pcr gives quantitative results. All persons with symptoms of tb disease, or either a positive tst or igra indicative of.
The pcr data were compared with conventional microscopy and culture technique and also with results from existing tb pcr kit. Materials and methods a total of 93 consecutive patients with hilarmediastinal lymphadenopathies. Polymerase chain reaction pcr is a method widely used in molecular biology to make several copies of a specific dna segment. Pcr can use the smallest sample of the dna to be cloned and amplify it to millions of copies in just a few hours. Pcr or polymerase chain reaction, is an amplification based molecular test, which under ideal laboratory conditions is a highly sensitive and specific method for the detection of target genes including those of mycobacterium tuberculosis the bacteria causing tuberculosis. The most commonly used techniques for tb diagnosis in. Genotyping guide description of genotyping methods. Pcr is a suitable technique for the differentiation of unrelated strains of h parasuis. The finding that the 12 field isolates of h parasuis all shared the same fingerprint is strong evidence that there was a common source of infection on all three farms. Tb dna in the cerebrospinal fluid csf through the use of various molecularbased methods, including nucleic acid amplification naa assay technique, particularly polymerase chain reaction pcr assay, has emerged as a promising new method for the diagnosis of cns tuberculosis because of its rapidity, sensitivity.
Tb test tuberculin skin test tst the tst has been in existence for over 100 years. Clinical evaluation of a mycobacterium tuberculosis pcr assay. Materials and methods a total of 93 consecutive patients with hilarmediastinal. Nucleic acid amplification assays including pcr have revolutionized the detection of mycobacterium tuberculosis mtb. Tb laboratory techniques for diagnosing tuberculosis.
Pcrbased rapid detection of mycobacterium tuberculosis in. Use of real time polymerase chain reaction for detection of m. A small amount of liquid containing tb proteins is injected into the lower part of the arm. Realtime pcr technology used for tb pcr is based on taqman chemistry.
The utility of polymerase chain reaction pcr in the. Tuberculosis infection, but only after tb disease is excluded by obtaining three specimens for afb smear and culture because old tb cannot be differentiated from active tb disease based on radiographic appearance alone. A pcr test based on insertion sequence is1081 was developed to detect mycobacterium tuberculosis complex organisms in the peripheral blood. This is a test to see if you have genital tract tuberculosis, i. Use of pcr method based on is6110 for detection of. A rapid and sensitive method is therefore needed for detection of mycobacterium tuberculosis in these paucibacillary tissue samples.
To address the low sensitivity of current tb diagnostics for afb smearnegative cases, we developed a simple and labelfree pathogen enrichment using homobifunctional imidoesters his using a microfluidic slim platform followed by conventional mycobacterium tuberculosis pcr to extract low amounts of pathogens from clinical samples 3. Pcr methods are therefore particularly valuable when amounts of rna are low, since the fact that pcr involves an amplification step means that it is more sensitive. Clinical evaluation of the polymerase chain reaction for the. Blood samples from 16 patients reporting at the district tb hospital, karnal, india, with localized. The conventional techniques used in tb diagnosis like afb acid fast bacilli smear microscopy lack sensitivity and the gold standard, culture test takes time. The most notable advantage of pcr tests is their rapid turnaround time and reliability for an early detection of eptb, which may have important implications for clinical management and tb control. Analysis of different primers used in the pcr method. Early diagnosis of tb is an essential step in effective management of the disease and is critical to reducing the number of new cases early diagnosis of tb requires the use of screening tests that are accurate, easily implemented, and costeffective. Based on taqman pcr, we designed a novel technique consisting of an internally. Ssp, typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. Using two mtbspecific pcr primer systems, we found 48 of 49 tuberculosis tb patients. A test based on multiplex polymerase chain reaction pcr targeting the 38 kda gene and is6110.
Rapid diagnosis of mediastinal tuberculosis with polymerase. Does polymerase chain reaction test confirm tuberculosis. An inhouse multiplex pcr test for the detection of. Though elisa is the most commonly used method for the detection of mycobacterial antigens and antibodies, it fails when there is low.
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