Nucleic Acid Amplification Technology Research Articles

Review on Application of Nucleic Acid Amplification Techniques in Pharmaceutical Products Analysis

The aim of this study was to describe application of molecular diagnostic tests based on nucleic acid amplification technologies (NAATs) in pharmaceutical product analysis. NAAT have become widely established in clinical microbiology laboratories in recent years, as well as in quality control (QC) laboratories for food testing, and lately introduced to the pharmaceutical QC laboratories. The number of available nucleic acid and gene amplification-based rapid microbiological methods has increased over the last few years, and for good reason. When compare to standard culture based methods, NAAT provide a rapid, an accurate and reliable means for detecting specific microorganisms of interest, especially in pharmaceutical dosage forms that are required to be free of objectionable or specified pharmacopeial organisms. This article discussed about the different types of nucleic acid amplification techniques and methods available to pharmaceutical microbiologists working on quality control of pharmaceutical products and develop some awareness among other pharmaceutical scientists. This review also highlights limitations of these methods applied in industrial setup.

Standardization of NAT for Blood-Borne Pathogens

Assays based on nucleic acid amplification technology (NAT) are increasingly used for screening of blood and for diagnosis or monitoring of patients. Both regulatory requirements for blood screening and international recommendations for the treatment of patients are based on common reference materials available globally for the standardization of NAT assays. World Health Organization International Standards (WHO ISs) and International Reference Panels (WHO IRPs) are primary reference materials. The characterization and manufacture of WHO reference materials as well as their evaluation is performed on behalf of the WHO by collaborating centers; their establishment is decided upon by the WHO Expert Committee on Biological Standardization (ECBS). The potency of the first WHO IS is defined by the ‘international unit' (IU) which should be maintained upon replacement of the IS. The IU, unlike copy number or genome equivalent, is defined by the IS with a physical existence, is available worldwide, and allows traceability and comparability of results. The anticipated use of WHO ISs is the calibration of secondary standards or the validation of essential assay features, e.g. limit of detection.

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results. However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis. In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids.

Current Trends in the detection of Acute HIV Infection among Blood Donors: Reliability of Pooled Nucleic Acid Amplification Technology and the Need for Population Specific Algorithms: A Systematic Review

We conducted a systematic review of quantitative studies that evaluated the accuracy of nucleic acid amplification testing technologies (NAAT) compared their cost effectiveness and evaluated minipool NAAT against individual donor testing NAAT. PubMed, Cochrane and Google Scholar were used to identify relevant peer‐reviewed journal articles published in English language between 1999 (when NAAT was introduced) and 2013. MeSH key words included: Human immunodeficiency virus OR HIV AND nucleic acid amplification techniques OR pooled NAAT AND blood donors. Additional filters include: minipool-NAAT and individual donor testing-NAAT. After screening for duplication and relevance, 50 out of 4,181 articles were selected. Thirty six (36) studies which included 5 review article, 5 retrospective cohort studies, 20 cross sectional studies, 2 statistical modeling’s, 2 national guidelines and 2 prospective cohort studies were further synthesized. Articles were further sub-divided into 8 groups based their focus area which includes: prevalence, clinical sensitivity of the assay, analytical sensitivity, test technology, testing algorithm, limit of detection and cost effectiveness. Four of six studies with pool sizes of 10 to 50 donor plasmas with standard centrifugation recorded a clinical sensitivity of 100% while the remaining two whose plasma pool size of 96 and128 had sensitivities of 92.3% and 95.3%, respectively. All four studies that focused on analytical sensitivity using different samples and controls, including cadaveric samples reported 100% analytical sensitivity using various pool sizes. We recommend minipool NAAT testing after running a third generation ELISA as highly sensitive and cost effective algorithm for low income countries.

Next generation blood tests for detecting tuberculosis at the point-of-care: Overcoming challenges in product development

To control tuberculosis (TB), it is necessary to detect the disease and provide effective treatment to avert its progression and to interrupt any onward transmission from infectious pulmonary cases. Traditional diagnostic methods rely on the detection of the Mycobacterium tuberculosis (MTB) bacilli in clinical samples such as sputum. Testing is usually done on patients that have been coughing for some time, when transmission is likely to have already occurred. The impact of the GeneXpert MTB/RIF assay has so far had a limited impact on TB control because of its high cost and limited accessibility. A number of follow-up nucleic acid amplification technologies are being evaluated, but they also rely on testing for the presence of the bacteria in sputum. Diagnosing extra-pulmonary disease and TB in children remains very difficult and tests are needed that detect all forms of the disease. For developing countries with a high burden of TB, technology is needed that is robust, affordable and will avoid diagnostic delay; the tests must be readily accessible to the community without waiting for patients to self-refer to a TB testing clinic. Compromises may be needed between accuracy and access where a rapid imperfect test that can be used to test people in their community will have more benefit than a more perfect test that is not accessible. Traditional serological tests have failed for TB because of the complexity of the disease, but by testing for multiple markers, sensitivity and specificity can be improved and it is possible to differentiate latent infection and active disease. Examples of new assays that have the potential to change TB control shall be provided. The challenge now is to develop, manufacture, and distribute detection platforms that are safe, robust and affordable. Recent studies have revealed the market for TB diagnostics to be fragmented and small. Consultation with manufacturers has revealed that access to finance, management of intellectual property, regulatory acceptance and in some countries the lack of distribution networks are significant barriers to innovation. The lack of financial incentives and expectation of poor returns on investment discourages commercial companies, and there is a need for the not-for-profit and academic sectors to expand their role in product development and delivery. Biologists and technologists must work with manufacturers and healthcare providers to overcome the economic and political barriers to product development, manufacture and distribution. To this end, guidelines and a ‘road map’ for taking new in vitro diagnostic medical devices to market have been developed to raise awareness and sensitize the TB research community for the need for innovation in product delivery.

Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA.

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.

Progress of improving blood donor screening by nucleic acid technology

With increasing application of blood transfusion, the research of side-effects such as transfusion-transmitted infections (TTIs) became more and more important. Up to the 90's of the 20th century, the first blood donor screening for pathogens transfected from blood transfusion entirely depended on serological test. At this time, the detection of virus were performed mainly by using method of detecting antibody, except hepatitis B virus (HBV) can be detected by hepatitis B surface antigen (HBsAg). Now, the molecular technologies, such as the polymerase chain reaction (PCR), have been used in clinic. These technologic methods can provide capability of detection for blood donor screening and reduced possibility of infection from blood transfusion. This review summarises the development of nucleic acid amplification technology and describes its current state.

Infectious risks of blood transfusions: Recent advances in testing technologies and new approaches to surveillance and decision‐making

IntroductionDespite impressive progress in reducing risk of established transfusion‐transmitted infectious diseases (TTIDs), new emerging infectious disease (EID) threats continue to be identified and cause pressure to expand deferrals and testing in order to maintain the safety of transfusions.AimsReview recent progress in developing approaches for estimation of residual risk and test efficacy for established TTIDs, as well as new approaches for systematic surveillance and assessment of EIDs that may threaten the blood supply.MethodsSummarize recent publications and initiatives of the global blood banking community directed at reducing risk of traditional TTIDs as well as recent responses to EID threats.ResultsImplementation of enhanced serological assays and nucleic acid amplification technologies (NAT) has virtually eliminated risks of major TTIDs. Formal approaches have recently been developed to monitor and respond to EIDs by examining their properties with respect to transmissibility and potential for causing significant disease in transfusion recipients. New tools for quantitative risk assessment are under development to facilitate rigorous evaluation of scientific evidence, while also formally soliciting and considering public perception and fear and health economic realities.ConclusionThese ongoing efforts at refining our risk surveillance, assessment and decision analysis tools, coupled with focused research to quantify risks and evaluate interventions, presage an era of sustained safety and enhanced public trust in the blood supply.

Usefulness of Pneumotest-Latex for Direct Serotyping of Streptococcus pneumoniae Isolates in Clinical Samples

This study evaluated the usefulness of the Pneumotest-Latex assay for serotyping Streptococcus pneumoniae isolates directly in clinical samples. With an agreement of 88.1% with a PCR-based reference method, this test can be a useful tool for this study purpose, especially in clinical laboratories that do not have access to nucleic acid amplification technologies.

Research and Implementation of Real Time Fluorescent Nucleic Acid Amplification Detection Technology

This paper introduces the research results of real-time detection of fluorescent nucleic acid amplification technology, mainly including the cooling rate, the precision of temperature control, temperature accuracy, module temperature uniformity, fluorescence intensity detection repeatability and measurement precision and the threshold cycle number. The working principle is based on the operating parameters of computer input, controlling module component temperature placed reagent sample changes, meeting the reagent sample high temperature denaturation, annealing temperature (complex), suitable temperature extension process cycle the reagent sample amplification, and through the photoelectric detection system according to the set of sample fluorescence changes. Analysis of data is processed by software. The results can be used for measurement of polymerase chain reaction and fluorescence quantitative examination of clinical diagnosis fields of immunology, the human genome engineering, forensic medicine, oncology, organization and population biology, paleontology, botany, animal science research field and virus, tumor, genetic diseases.

Molecular diagnosis in clinical parasitology: When and why?

Microscopic detection and morphological identification of parasites from clinical specimens are the gold standards for the laboratory diagnosis of parasitic infections. The limitations of such diagnostic assays include insufficient sensitivity and operator dependence. Immunoassays for parasitic antigens are not available for most parasitic infections and have not significantly improved the sensitivity of laboratory detection. Advances in molecular detection by nucleic acid amplification may improve the detection in asymptomatic infections with low parasitic burden. Rapidly accumulating genomic data on parasites allow the design of polymerase chain reaction (PCR) primers directed towards multi-copy gene targets, such as the ribosomal and mitochondrial genes, which further improve the sensitivity. Parasitic cell or its free circulating parasitic DNA can be shed from parasites into blood and excreta which may allow its detection without the whole parasite being present within the portion of clinical sample used for DNA extraction. Multiplex nucleic acid amplification technology allows the simultaneous detection of many parasitic species within a single clinical specimen. In addition to improved sensitivity, nucleic acid amplification with sequencing can help to differentiate different parasitic species at different stages with similar morphology, detect and speciate parasites from fixed histopathological sections and identify anti-parasitic drug resistance. The use of consensus primer and PCR sequencing may even help to identify novel parasitic species. The key limitation of molecular detection is the technological expertise and expense which are usually lacking in the field setting at highly endemic areas. However, such tests can be useful for screening important parasitic infections in asymptomatic patients, donors or recipients coming from endemic areas in the settings of transfusion service or tertiary institutions with transplantation service. Such tests can also be used for monitoring these recipients or highly immunosuppressed patients, so that early preemptive treatment can be given for reactivated parasitic infections while the parasitic burden is still low.

Validation of the ANSR Salmonella method for detection of Salmonella spp. in a variety of foods. Performance Tested Method 061203.

This study represents a proposal to extend the matrix claims for the ANSR Salmonella test, Performance Tested Method 061203. The test is based on the nicking enzyme amplification reaction (NEAR) isothermal nucleic acid amplification technology. The assay platform features simple instrumentation, minimal labor, and following a single-step 16-24 h enrichment (depending on sample type), an extremely short assay time of 30 min including sample preparation. Detection is real-time using fluorescent molecular beacon probes. ANSR Salmonella was originally validated for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, and oat cereal, and on stainless steel, plastic, sealed concrete, ceramic tile, and rubber surfaces. The matrixes tested in this study include pet food, ice cream, soy flour, raw almonds, peanut butter, spinach, black pepper, raw frozen shrimp, cocoa powder, and pasteurized dried egg. In unpaired comparative testing there were no statistically significant differences in the number of positive results obtained with the ANSR and the reference culture methods. Enrichment for 16 h was effective for all commodities tested except ice cream, black pepper, dried pasteurized egg, and 375 g samples of dry pet food, for which enrichment for 24 h is indicated.

A new ultrasonic high-throughput instrument for rapid DNA release from microorganisms

Rapid detection methods for microbiological contamination are requested by many industries that respond to public health concerns. The control laboratories are replacing traditional culturing methods with faster assays based on nucleic acid amplification technologies, such as real-time PCR. However, an optimal nucleic acid sample preparation method is critical for the sensitivity and specificity of such tools.A high-throughput automated external ultrasonic device was developed for rapid lysing of microorganisms. Based on Ct values obtained from real-time PCR, there was efficient DNA release from the 16 microorganisms tested, including Gram positive and negative bacteria, bacterial spores, yeasts and spores of molds. Linearity of the lysis method was also demonstrated for Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Candida albicans and the spores of Aspergillus brasiliensis, with correlation coefficients (r2) between 0.90 and 0.98. After sonication, PCR analysis of the lysates revealed earlier Ct values (0.83 to 7.49) for S. aureus, P. aeruginosa and S. enterica compared to the bead-beating method of lysis. These results demonstrated more efficient DNA release from bacteria with the ultrasonication system. Nevertheless, for fungi, the Ct values were 0.94 to 1.61 later for sonication than for bead beating.This study demonstrates that 4min of sonication with this new automated high-throughput instrument allows for the efficient lysis of a large range of microorganisms.

Application of Nucleic Acid Amplification Technology in Screening Blood Donors in Yancheng of Jiangsu Province

Objective To explore the feasibility and necessity of the application of nucleic acid amplification technology (NAT) in screening blood donors in Yancheng.Methods From July 4th,2011 to July 3rd,2012,a total of 34 925 blood samples which were negative in enzyme-linked immunosorbent assay (ELISA) and qualified in alanine aminotransferase (ALT) detection,were sele.cted in this study stochastically.All the samples were detected hepatitis B virus (HBV)/ hepatitis C virus / human immunodeficiency virus by Roche Cobas S201 System in 6 samples pooling.NAT positive samples were reconfirmed by National Center for Clinical Laboratories (NCCL).Results The results showed that 57 samples in 34 925 serological negative specimens were NAT positive,and the rate of positive detection was 0.16 ％.Twenty of them were reconfirmed by NCCL and 13 were conformed HBV positive presented a viral load below 20 IU/mL.Of the 13 reconfirmed HBV positive samples,9 samples were occult hepatitis B (OBI),2 samples were ELISA omission.Conclusions ELISA could miss some virus,and occult infection of HBV are exist in local blood donors.NAT could be the supplement to the ELISA to improve the safety of local clinical blood use. Key words: Hepatitis B virus; Hepatitis C virus; Human immunodeficiency virus; Nucleic acid amplification technology

Getting Things Backwards to Prevent Primer Dimers

This Commentary highlights the article by Satterfield that describes a new class of primer technology-cooperative primers, which prevent primer-dimer amplification.

Multi-Detection by Target Mixed Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP), a nucleic acid amplification technology, has been characterized as having a short reaction time while generating a large amount of DNA product under isothermal conditions. Here, we found that LAMP is able to amplify more than one target simultaneously by using a mixture of human immunodeficiency virus1, hepatitis B virus, and hepatitis C virus primers, a process we have named target mixed LAMP. Target mixed LAMP and detection can be performed in less than 1 h. In this study, we also used target mixed LAMP to successfully amplify GAPDH, gamma-actin, TATA box binding protein, and haptoglobin from pooled cDNA derived from mouse tissues. Furthermore, using eight plasmid vectors as templates, target mixed LAMP is able to amplify all targets simultaneously. These findings reveal the usefulness of LAMP for the diagnosis of infectious disease as well as for conducting gene expression analysis.

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity, cost-effectiveness and their suitability for high-throughput testing. Enzyme immunoassay (EIAs) for hepatitis C virus (HCV) antibodies were the initial screening tests introduced. The "first generation" antibody EIAs detected seroconversion after unduly long infectious window period. Improved HCV antibody assays still had an infectious window period around 66 d. HCV core antigen EIAs shortened the window period considerably, but high costs did not lead to widespread acceptance. A fourth-generation HCV antigen and antibody assay (combination EIA) is more convenient as two infectious markers of HCV are detected in the same assay. Molecular testing for HCV-RNA utilizing nucleic acid amplification technology (NAT) is the most sensitive assay and shortens the window period to only 4 d. Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now < 1 per million donations. However, HCV serology still continues to be retained as some donations are serology positive but NAT negative. In resource constrained countries HCV screening is highly variable, depending upon infrastructure, trained manpower and financial resource. Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres. The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays. Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.

Can HCV core ag testing replace the routine screening assays in patients on hemodialysis?

Dear Editor We have recently read with great interest the article published in your most valuable journal entitled “Hepatitis C virus infection rate among seronegative hemodialysis patients screened by two methods: HCV core antigen and polymerase chain reaction” by Moini et al. (1). This study focused its message on determination of the prevalence of hepatitis C virus (HCV) infection in seronegative hemodialysis (HD) patients using two methods of HCV core antigen (Ag) testing by ELISA assay, and the real time polymerase chain reaction. The authors have concluded that HCV core Ag testing could be used as a sensitive method for HCV infection screening in patients on HD (1). We agree that patients on long-term HD are at higher risk of acquiring HCV infection (2, 3). Despite administration of recombinant erythropoietin, HCV transmission is still being reported among patients on HD (4, 5). Undoubtedly, HCV screening is crucial for patients on HD since these population is at increased risk of infection and moreover it easily spreads the infection through dialysis units (6). HCV infection is routinely diagnosed by measuring anti-HCV antibody whereas the antibody is undetectable in the first four to six weeks of infection, the so-called, window period (7). Furthermore, there is a blunted antibody response in patients on HD due to depressed immune system (1). Kidney Disease Improving Global Outcomes (KDIGO) has recommended the use of nucleic acid amplification technology (NAT) in detection and evaluation of HCV in chronic kidney disease (8). By utilizing NAT, some problems emerge: the kits are frequently unavailable; considerable skill is required; there is limited reproducibility; and most importantly, the costs are steep (7). Some studies in the general population have pointed out the importance of HCV core Ag detection as an alternative to NAT for early diagnosis of infection as well as predicting and monitoring the response to therapy (9); however few studies concerning its efficacy in patients on long-term HD exist (7, 10). We totally concur with the authors that HCV core Ag may be an accurate marker for early detection and virological monitoring of HCV infection in patients on HD. Owing to the low cost and its technical easiness, this assay can be employed for routine screening of patients on HD; however, more studies with larger sample size are still required to effectively establish HCV core Ag test as a preferred assay for the early diagnosis of HCV infection in patients on HD.