Nucleic Acid Amplification Technology Research Articles

Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

BackgroundSignal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus.ResultsHere we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20) was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis.ConclusionThe SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

P1416 Statistical modelling of the performance of nucleic acid amplification technologies in clinical diagnostic applied to quality control for molecular diagnostics hepatitis B virus programmes

P1416 Statistical modelling of the performance of nucleic acid amplification technologies in clinical diagnostic applied to quality control for molecular diagnostics hepatitis B virus programmes

69. LABORATORY CONTRIBUTION FOR CONTROL OF CHLAMYDIA

Infection with Chlamydia trachomatis may present a wide spectrum of clinical symptoms in patients. Diagnosis based on presenting symptoms however may be of limited value in asymptomatic patients. Therefore, laboratory tests are utilized; in particular nucleic acid amplification technology (NAAT) based assays. In the recent years, NAAT testing has improved diagnosis of chlamydia to a great degree. The increased sensitivity of NAAT assays has also allowed for better detection of chlamydia in extragenital samples, diagnosis of lymphogranuloma venereum and has made the use of non-invasive self-collected samples possible. Chlamydia genotyping and sequence based comparisons are also increasingly being used in investigation of types present in the population as well as providing possibility of differentiating a new infection from re-infection. Such methods continue to play a major role not only in patient diagnosis but also in epidemiology and public health.

Lyophilized standards for the calibration of real time PCR assay for hepatitis C virus RNA

Since October 1997, an international standard for hepatitis C virus (HCV) nucleic acid amplification technology assay, 96/790, has been available. We compared a series of lyophilized standards with known HCV RNA concentrations against the international standard in fluorescence quantitative PCR detection. A series of lyophilized sera were calibrated by ROCHE COBAS AMPLICOR HCV Monitor test against the international standard and sent to various manufacturers to analyse the samples using their own kits. Then calibration curves from the series were compared with that obtained from the external standard calibration curve with the manufacture's series. The standard calibration curve with the series of lyophilized serum showed an excellent correlation (R(2) > 0.98), slope and intercept that were similar to those from the manufacture's series. When the standard calibration curve from the series of lyophilized standards were used to define the values of the given sample, lower coefficients of variation between kits from different manufactures were obtained. The results showed that the lyophilized standards could be used to setup the standard calibration curve for clinical HCV RNA quantitative PCR detection.

SI19 NAT or Not to NAT – This is the Dilemma?

The National Blood Service has undertaken a strategic review of Nucleic acid Amplification Technology (NAT) Testing used to screen blood for Hepatitis C Virus (HCV) at the request of the Department of Health.Three strategic options have been considered and are presented within this review:Continue testing (which in practice also includes developing this technology further as new automated platforms and multiplex tests become available).Substitute HCV antigen testing for NAT testingStop NAT HCV testingIn essence, this review concludes that NAT HCV testing is an extremely expensive intervention. Now that NAT testing is well developed by Blood Services world‐wide and has been in use to screen the UK population for some time, the consequences of pursuing each of the options presented can be predicted with accuracy.

Clinical assessment of assays for diagnosis of herpes simplex infection

It is becoming increasingly clear that the herpes simplex viruses (HSVs) 1 and 2 constitute a major, global, public health problem, particularly as genital herpes is implicated in the causation of a significant percentage of onwards transmission of the HIV virus. A major factor in the transmission of HSV is that most carriers are unaware of their diagnosis. In the last few years, the development of nucleic acid amplification technology and type-specific antibody serology to test for HSV-1 and -2 has contributed significantly to the accurate diagnosis of these infections. Despite guidance to the contrary, there is still much use of less sensitive tests such as viral culture and antibody testing based on crude antigen. It is essential that we use the most sensitive and specific diagnostic tests if we are to curb this epidemic.

Anti‐HBc screening of blood donors: a comparison of nine anti‐HBc tests

Since voluntary introduction of hepatitis B virus (HBV) minipool nucleic acid amplification technology (NAT) at the German Red Cross, the expected residual risk of a transfusion-associated HBV infection has been estimated to be 1 : 500,000 - about 10 times higher than for human immunodeficiency virus (HIV) or hepatitis C virus (HCV) infection. Donors demonstrating chronic positivity for antibody to hepatitis B core antigen (anti-HBc), negativity for hepatitis B surface antigen (HBsAg) and polymerase chain reaction (PCR)-negative with a low virus load are a major cause of this increased risk. Ten-thousand blood donors from our blood-donation centre were screened for anti-HBc using the current PRISM HBc and the new PRISM HBcore assay to evaluate the diagnostic sensitivity and specificity of these tests. PRISM HBc- or PRISM HBcore-reactive samples were further analysed using seven additional tests for anti-HBc, two tests for antibody to hepatitis B surface antigen (anti-HBs), one test for antibody to hepatitis B envelope antigen (anti-HBe) and three HBV NAT assays. From a total of 10,000 donors, nine and 14 samples were reactive only in the PRISM HBc and the PRISM HBcore, respectively, whereas 165 samples were reactive in both anti-HBc assays. Further analysis of these 188 anti-HBc-reactive specimens in a total of nine different anti-HBc assays revealed concordant results for 162 (86.2%) specimens. Sample cut-off values for anti-HBc were significantly (P < 0.01) lower for anti-HBc-only reactive samples compared with specimens that were also reactive for anti-HBs or anti-HBe. Both PRISM anti-HBc assays revealed that approximately 1.8% of non-prescreened blood donors from Germany were reactive for anti-HBc. Although sensitivity was comparable between both assays, specificity was increased significantly with the PRISM HBcore. High anti-HBc sample cut-off values were indicative for reactivity in other HBV parameters and for concordant results in the nine different anti-HBc assays. Look-back investigations are necessary to estimate the infection risk both of anti-HBc-only positive and of anti-HBc/anti-HBs-positive blood transfusions.

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.

Multicenter proficiency study for detection of Toxoplasma gondii in amniotic fluid by nucleic acid amplification methods

A proficiency panel was designed to assess the performance of nucleic acid amplification technologies for the detection of Toxoplasma gondii in amniotic fluid. Methods The proficiency panel consisted of five lyophilised coded samples in a range of concentration between 5 to 1000 parasites/ml and a negative control. The distribution also included a questionnaire on the applied methods. Results Thirty-three laboratories in 17 countries participated and returned a total of 38 data sets. The percentage of data sets achieving correct results on all panel samples was 42.1%, whereas two or more incorrect or equivocal results were reported for 36.8%. The lowest concentration (5 parasites/ml) was not identified correctly in 15 (39.5%) data sets. False positive results were reported by two laboratories both of which had not included a step in their procedure to rule out contamination. In 32 (84.2%) data sets an “in-house” method was used, and in 6 (15.8%) sets a commercial assay was applied. Conclusions Overall, the results of this study demonstrate the need for improvements in both sensitivity and specificity of molecular detection methods of T. gondii and for the development of international reference materials to help laboratories with the development and validation of their assays.

Establishment of the first national standard for nucleic acid amplification technology (NAT) assay for HCV RNA

目的 研制国内用于丙型肝炎病毒核酸(HCV RNA)扩增检测的血清标准物质.方法用HCV RNA阴性血浆将阳性血浆稀释至含量约300 000拷贝数/ml,然后进行真空冷冻干燥.检测方法采用实时荧光定量聚合酶链反应方法和罗氏公司的PCR内标定量方法.制备标准品含量通过与国际标准(世界卫生组织属下的国家生物标准研究所标准物质编号:96/790)比对获得.结果该标准物质定值为:(1.29±0.24)×105 IU/ml.其稳定性实验表明室温20～25℃(相对湿度20%～50%)14 d、37℃(相对湿度60%～80%)7 d均稳定.长期监测结果表明: 2～8℃6个月及-20℃保存两年的含量均无明显变化.均一性检测结果:瓶间不精密度为3.53%.结论该制备物可以作为用于核酸扩增检测的丙型肝炎病毒核酸国家标准物质。

Genetics-Based Methods for Detection of Salmonella spp. in Foods

Genetic methods are now at the forefront of foodborne pathogen testing. The sensitivity, specificity, and inclusivity advantages offered by deoxyribonucleic acid (DNA) probe technology have driven an intense effort in methods development over the past 20 years. DNA probe-based methods for Salmonella spp. and other pathogens have progressed from time-consuming procedures involving the use of radioisotopes to simple, high throughput, automated assays. The analytical sensitivity of nucleic acid amplification technology has facilitated a reduction in analysis time by allowing enriched samples to be tested for previously undetectable quantities of analyte. This article will trace the evolution of the development of genetic methods for detection of Salmonella in foods, review the basic assay formats and their advantages and limitations, and discuss method performance characteristics and considerations for selection of methods.

Nucleic acid amplification technology for screening of HBV DNA among anti‐HBc‐positive units of blood and blood donors

Nucleic acid amplification technology for screening of HBV DNA among anti‐HBc‐positive units of blood and blood donors

Hepatitis B virus genotype G monoinfection and its transmission by blood components

An acute hepatitis B virus (HBV) infection was diagnosed in a regular apheresis (plasma/platelet) donor by the hepatitis B surface antigen (HBsAg) assay and minipool nucleic acid amplification technology (NAT). The acute infection was confirmed by detection of anti-HBc (IgM) and anti-HBs 2 weeks later. The donor showed no clinical symptoms and had normal alanine aminotransferase levels. He had a history of weekly apheresis plasma or platelet donations. Archived material from the donor and the respective recipients was investigated by sensitive HBV NATs as part of a look-back procedure. HBV DNA was detectable in previous donations as well as in two recipients transfused with platelet concentrates. The rare HBV genotype G was identified in all HBV-DNA-positive samples. Strong evidence of genotype G monoinfection was obtained by clonal sequencing, HBV genotype line probe assay, genotype-specific NATs, and restriction pattern analysis. In contrast to previously described genotype G infections, which all appeared as coinfections with genotype A, neither the hepatitis B e antigen (HBeAg) nor anti-HBe was detectable in any of the samples. This shows that HBeAg is dispensable for viral replication. The delay in detecting HBsAg in both the donor and recipient samples may be explained by either decreased genotype G-specific synthesis of incomplete viral forms in early HBV infection or the lower sensitivity to genotype G of the current HBsAg assays. In conclusion, this reported case of an HBV infection was caused exclusively by genotype G.

Strand displacement amplification for Chlamydia trachomatis detection in rectal pharyngeal and eye swabs

Nucleic acid amplification procedures can be used to overcome the limitations of direct probe hybridization assays. A number of such systems have been described. Each method makes use of in vivo mechanisms of nucleic acid replication and repair to effect the amplification of nucleic acids in vitro. The strength of nucleic acid amplification technology, the ability to make many copies of a sequence present initially at a low concentration, is also its greatest potential weakness. Carryover contamination of amplification products represents a great risk for false positivity in these assay systems. Various methods have been developed to reduce the contamination risk, allowing the adaptation of amplified probe assays for routine use in the clinical laboratory.

Valutazione di controlli di qualità per sistemi di amplificazione molecolare qualitativi e quantitativi nella ricerca di HBV-DNA, HCV-RNA, CMV-DNA, e HIV-DNA

NAT (Nucleic Acid Amplification Technology) assays are now widely used in diagnostic microbiology. The implementation of NAT assays requires the use and the availability of control reagents, with various aims, such as to verify the efficiency of the procedures and the comparability of the results obtained among different laboratories. At this purpose, the Committee for the Study of Biotechnologies (CoSBio) of AMCLI performed a study to evaluate a group of synthetic quality controls for the detection of HBV-DNA, HCV-RNA, HIV-DNA e CMV-DNA, commercially distributed by AlfaWassermann. Here we report the results of the first part of the study performed to assess the main characteristics of these reagents and their applicability to different diagnostic systems. In the second part, the same controls will be evaluated in a national multicentre study.

HIV-1 NAT minipool during the pre-seroconversion window period: detection of a repeat blood donor

The introduction of nucleic acid amplification technology (NAT) for screening pooled or individual donations remarkably improved the safety of blood products. The size of mini-pooled NAT is considered critical for identification of HIV-1 infected donors during preseroconversion phase of infection. We describe a case of HIV-1 infection in a serologically negative repeat blood donor identified by 16 minipool (MP) NAT. The donation was tested by Roche Cobas AmpliScreen HIV-1 Test with manual extraction (MultiPrep Specimen Processing Procedure). The sensitivity of different MP sizes was observed. Serial samples of infected donor were examined with different third and fourth generation HIV-1 serological assays. In the index donation viral load was 515 copies/ml corresponding to about 50 IU when diluted in 16 MP. Abbott third and fourth generation EIA tests detected the seroconversion four days later the index donation. The report emphasizes the relevance of a very small size of MP to really reduce the window serologic phase of current EIA test by HIV-1 NAT test.

Prevalence of Parvovirus B19 and Hepatitis A virus in Portuguese blood donors

Introduction In recent years, concern about the safety of blood in regard to the transmission of blood-borne viruses has been decreased. Safety has been achieved with a combination of different strategies, such as careful selection of donors, screening for relevant virological markers and viral inactivation/removal methods. More recently, the implementation of the nucleic acid amplification technologies for the detection of HIV-1, HCV and HBV, has increased safety by reducing the “window period” of the infections. Other viruses, such as Parvovirus B19 (PB19) and Hepatitis A virus (HAV), can cause problems for blood safety. These infections could provoke serious complications in some risk groups, such as pregnant women, patients with hematological problems, children and patients with immunodeficiencies. Materials and methods An observational study was performed to determine the prevalence of PB19 and HAV in Portuguese blood donors. We gathered, during four months, 5025 plasma donations and made them into 505 pools with no more than 10 donations each. The nucleic acids were isolated using MagNA Pure LC (Roche, Mannheim, Germany). A “Real Time PCR” (LightCycler, Roche) was used to perform the nucleic acid amplification and detection, using kits from the manufacturer. Results We found a prevalence of 0.12% for PB19 and 0% for HAV. Viraemia levels found in the positive donations range from 7.1 × 10 4 to 2.1 × 10 12 IU/ml. Discussion This study demonstrates the possibility of performing these tests in routine blood banks. We found a similar prevalence of PB19 when compared with other European and USA countries. In the case of HAV, we predict a maximum risk of 0.06% for a donor to be infected. It is necessary to perform other studies, including cost/benefit analysis to evaluate the risks and profits of implementing these methodologies in Transfusion Medicine.

Challenges in the Testing of Non-Heart-Beating Cadavers for Viral Markers: Implications for the Safety of Tissue Donors

Natural changes that occur in blood and tissue after death may result in false positive results in antigen and antibody detection tests performed to identify markers of viral infection in potential tissue donors. Such tissue, which might otherwise be acceptable for therapeutic purposes, would not meet current standards for safe tissue banking. This is especially important in the context of insufficiency in the tissue supply. In this study, a series of blood samples collected during routine post-mortem examination was assayed using a range of commercially available kits for the detection of HBsAg, anti-HCV and anti-HIV 1 + 2 antibody/antigen. Results of tests on 104 samples collected from 97 individuals indicate that some kits result in a higher number of initial reactive samples than others. Approximately 40% of samples were reactive in one or more HBsAg assay, less than 10% in at least one anti-HIV kit and only 1 sample at low level on an anti-HCV kit. Liver or lymph node samples from individuals whose serum sample gave reactive results in antigen/antibody assays were tested for viral nucleic acid in the corresponding nucleic acid amplification test. Only one individual's sample was confirmed to test positive for HBsAg in a confirmatory neutralisation test and by nucleic acid amplification technology, and a second individual whose serum was scored reactive for anti-HCV, but negative for HBsAg, had a liver sample which was HBV DNA positive and HCV RNA negative. The results of the study indicate that antibody/antigen assays are not as specific as NAT using state of the art DNA extraction techniques. Both types of assay complement each other and used together will help assure the safety of tissues for transplantation.

Sexually transmitted infection and blood-borne virus screening in juvenile correctional facilities: A review of the literature and recommendations for Australian centres

Background Juveniles in custody are disproportionately affected by sexually transmitted infections (STI) and blood-borne viruses (BBV) due to high rates of risk behaviours. Methods A literature review was undertaken with the aim of providing evidence-based recommendations on STI/BBV screening in Australian juvenile correctional facilities. Relevant research was identified using Premedline and Medline databases, followed by a manual search of reference lists in relevant articles identified in the database search. A total of 36 relevant publications were identified and reviewed. Results STI/BBV knowledge in incarcerated youth is poor and accompanied by high rates of sexual and blood-borne risk behaviours. The prevalence of these infections is considerable. High rates of asymptomatic gonococcal and chlamydial infections exist, which can be easily diagnosed on self-collected specimens using new nucleic acid amplification technology. HIV infections are rare although continued vigilance is needed in view of substantial risk factors for infection. Hepatitis C prevalence is high, although much lower than that of adult prisoners, signifying a possible window of opportunity for Hepatitis C prevention. Many remain at risk of Hepatitis B, and it is important to assess the need for vaccination in this group. Conclusion Screening for STI/BBV in incarcerated juveniles is of major public health importance and all individuals should be offered screening in conjunction with risk-reduction education during their admission to juvenile detention centres.

Evaluation of an automated high‐volume extraction method for viral nucleic acids in comparison to a manual procedure with preceding enrichment

Nucleic acid extraction still harbours the potential for improvements in automation and sensitivity of nucleic acid amplification technology (NAT) testing. This study evaluates the feasibility of a novel automated high-volume extraction protocol for NAT minipool testing in a blood bank setting. The chemagic Viral DNA/RNA Kit special for automated purification of viral nucleic acids from 9.6 ml of plasma by using the chemagic Magnetic Separation Module I was investigated. Analytical sensitivity for hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), hepatitis A virus (HAV) and parvovirus B19 (B19) was compared to our present manual procedure that involves virus enrichment by centrifugation. Chemagic technology allows automation of the viral DNA/RNA extraction process. Viral nucleic acids were bound directly to magnetic beads from 9.6-ml minipools. By combining the automated magnetic beads-based extraction technology with our in-house TaqMan polymerase chain reaction (PCR) assays, 95% detection limits were 280 IU/ml for HCV, 4955 IU/ml for HIV-1, 249 IU/ml for HBV, 462 IU/ml for HAV and 460 IU/ml for B19, calculated for an individual donation in a pool of 96 donors. The detection limits of our present method were 460 IU/ml for HCV, 879 IU/ml for HIV-1, 90 IU/ml for HBV, 203 IU/ml for HAV and 314 IU/ml for B19. The 95% detection limits obtained by using the chemagic method were within the regulatory requirements for blood donor screening. The sensitivities detected for HCV, HBV, HAV and B19 were found to be in a range similar to that of the manual purification method. Sensitivity for HIV-1, however, was found to be inferior for the chemagic method in this study.