Nucleic Acid Amplification Technology Research Articles

Rethinking Approaches to Improve the Utilization of Nucleic Acid Amplification Tests for Detection and Characterization of Influenza A in Diagnostic and Reference Laboratories

Influenza A virus (IFVA) is a significant cause of respiratory infections worldwide and was also responsible for a recent pandemic in 2009. Laboratory identification of IFVA can guide antiviral therapy, assist in cohorting of patients and prevent antibiotic use. Characterization of the virus can track the emergence of novel strains, identify resistance and determine how circulating strains match with vaccine components. The gold standard for detection and characterization of IFVA is nucleic acid amplification technology (e.g., reverse transcriptase PCR [RT-PCR]), which must contend with a constantly evolving viral genome. Although molecular technology has been available for over two decades, there is still an operational gap between assay design and utilization of these tests for the diagnosis and characterization of IFVA. This review will discuss issues surrounding the implementation and use of RT-PCR for the identification and characterization of IFVA, and speculate on why RT-PCR has not been used more widely in clinical laboratories or moved closer to the patient. Newer, less widely used technologies that may change our laboratory practices will be identified and the authors will close with an attempt to identify some future applications of RT-PCR-based technologies for the detection and characterization of IFVA.

Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing.

Collaborative study to establish a World Health Organization International genotype panel for parvovirus B19 DNA nucleic acid amplification technology (NAT)‐based assays

The aim of the collaborative study was to evaluate a panel of plasma samples containing different genotypes of parvovirus B19 (B19V) for use in nucleic acid amplification technology (NAT)-based assays. The panel of samples [Center for Biologics Evaluation and Research Parvovirus B19 Genotype Panel 1; National Institute for Biological Standards and Control (NIBSC) code number 09/110] comprises four different members, i.e. Member 1, Member 2, Member 3, and Member 4 (M1-M4); these represent genotypes 1, 2, 3a B19V, and a negative plasma control, respectively. Thirty-five laboratories from 13 different countries participated in the study. Participants assayed the panel members concurrently with the 2nd World Health Organization (WHO) International Standard for B19V DNA (NIBSC code 99/802) on four separate occasions. A total of 44 sets of data were returned, 34 from quantitative assays and 10 from qualitative assays. The majority of assays used were in-house and based on real-time PCR. The results showed that all three genotypes were detected consistently by the majority of participants, although a small number of assays detected genotypes 2 and 3 less efficiently, or not at all. Real-time stability studies have indicated that the panel of B19V samples is stable under normal conditions of storage, i.e. ≤-70°C. The four-member panel is intended for use in evaluating the ability of NAT assays to detect different B19V genotypes (M1-M3). Based on the results of the collaborative study, the panel was established as the 1st WHO International Reference Panel for parvovirus B19 genotypes.

Relative distribution of West Nile virus RNA in blood compartments: implications for blood donor nucleic acid amplification technology screening

Despite implementation of targeted individual-donor nucleic acid test (NAT) screening of blood donors for West Nile virus (WNV), three "breakthrough" WNV transfusion transmission cases were reported (2004-2008), suggesting that current plasma-based assays are unable to detect all WNV-infectious donations. A 2007 report found that 19 of 20 red blood cell components from WNV-infected donors contained 1 log higher viral load than plasma components. This study's aim was to further establish the value of screening whole blood relative to plasma for WNV RNA by generating differential viral loads on paired samples derived from blood screening tubes. WNV RNA-positive donors identified by routine NAT screening were enrolled and quantitative viral data were generated using cross-sectional (index-donation) and longitudinal (follow-up) specimens. A real-time reverse transcription-polymerase chain reaction viral load assay was used on both study sample sets and replicate qualitative NAT screening assays were also used on the longitudinal study samples. For the cross-sectional study, seronegative index donations (n = 29) had WNV RNA concentrations fourfold higher in plasma than in whole blood, whereas for seropositive donations (n = 13), the WNV RNA concentrations were 10-fold higher in whole blood than in plasma. All 10 longitudinal study participants were seropositive throughout the follow-up study; whole blood viral load was consistently greater than plasma viral load (mean difference, 343 copies; p < 0.001) up to 200 days after index. The improved sensitivity of WNV NAT using whole blood instead of plasma was confirmed, but appears to be limited to better detection in seropositive stages. However, the implication of these findings for blood screening requires further study to establish the infectivity of persistent whole blood viremia.

S2.4 New technologies to support STI screening and syndromic management in resource-limited settings

BackgroundHigh quality tests for detection of STIs are available but they are neither affordable nor available to patients in resource-limited settings. For patients presenting with STI symptoms, WHO recommends the...

No viremia of pandemic (H1N1) 2009 was demonstrated in blood donors who had donated blood during the probable incubation period

In the spring of 2009, the novel swine-origin influenza A (pandemic [H1N1] 2009) virus emerged and spread globally. Although no established cases of transfusion-transmitted influenza have been reported, the widespread outbreak of pandemic (H1N1) 2009 caused serious concern regarding the safety of blood products. The Japanese Red Cross Blood Centers have intercepted blood products with accompanying postdonation information indicating possible pandemic (H1N1) 2009 infection. To study the risk of transmission of pandemic (H1N1) 2009 by blood transfusion, we searched for the viral genome in such products using nucleic acid amplification technology. Between June and December 2009, blood components were collected from 579 blood donors who were diagnosed as or strongly suspected of having pandemic (H1N1) 2009 within 7 days after donation. Viral RNA was extracted from plasma and red blood cell (RBC) products, and RNA samples were subjected to real-time reverse transcription-polymerase chain reaction of the hemagglutinin and matrix genes of the pandemic (H1N1) 2009 virus. A total of 565 plasma and 413 RBC products from the 579 blood donors were available. No viral RNA of the pandemic (H1N1) 2009 was detected in any of the blood samples from the 579 blood donors. No viremia of pandemic (H1N1) 2009 was demonstrated in any of the 579 blood donors who had most likely donated blood during the incubation period. It is considered that the risk of transmitting pandemic (H1N1) 2009 by blood transfusion is extremely low.

The prevalence of human parvovirus B19 DNA and antibodies in blood donors from four Chinese blood centers

Human parvovirus B19 is a common human pathogen that causes a variety of diseases with outcomes ranging from asymptomatic to severe, especially in immunocompromised patients. The B19 virus can be transmitted via blood and/or blood products and its resistance to common viral inactivation and/or removal methods raises the importance of B19-related blood safety. However, the existence, variation, and loading of B19 in Chinese blood donors have not been determined. Quantitative polymerase chain reaction (PCR) was developed to detect all three genotypes of the human erythrovirus DNA in plasma samples. In total, 3957 donations from four Chinese blood centers were screened for B19 by real-time minipool nucleic acid amplification technology (NAT). The positive samples were then confirmed by nested PCR and subjected to sequence analysis and alignment for phylogenetic studies. An enzyme-linked immunosorbent assay-based experiment was also performed to identify the prevalence of immunoglobulin (Ig)G and/or IgM antibodies specific to the B19 structural proteins in acquired samples. Of 3957 blood donors, 23 (0.58%) specimens were found positive for B19 DNA. The quantitative DNA levels ranged from 2.48 × 10(2) to 6.38 × 10(4) copies/mL. The phylogenic analyses showed that the prevalent genotypes in Chinese blood donors belong to B19 Genotype 1. A total of 448 samples from Chinese blood donors were investigated for the seroprevalence of B19 antibodies, among which 24.6 and 6.9% of specimens were seropositive for B19 IgG and IgM antibodies, respectively. A total of 2.5% of these samples were positive for both antibody isotypes. Whether B19 NAT screening of blood and blood products should be launched in China, larger studies are needed to facilitate an informed decision.

A self-heating cartridge for molecular diagnostics

A disposable, water-activated, self-heating, easy-to-use, polymeric cartridge for isothermal nucleic acid amplification and visual fluorescent detection of the amplification products is described. The device is self-contained and does not require any special instruments to operate. The cartridge integrates chemical, water-triggered, exothermic heating with temperature regulation facilitated with a phase-change material (PCM) and isothermal nucleic acid amplification. The water flows into the exothermic reactor by wicking through a porous paper. The porous paper's characteristics control the rate of water supply, which in turn controls the rate of exothermic reaction. The PCM material enables the cartridge to maintain a desired temperature independent of ambient temperatures in the range between 20 °C and 40 °C. The utility of the cartridge is demonstrated by amplifying and detecting Escherichia coli DNA with loop mediated isothermal amplification (LAMP). The device can detect consistently as few as 10 target molecules in the sample. With proper modifications, the cartridge also can work with other isothermal nucleic acid amplification technologies for detecting nucleic acids associated with various pathogens borne in blood, saliva, urine, and other body fluids as well as in water and food. The device is suitable for use at home, in the field, and in poor-resource settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent.

World Health Organization collaborative study to calibrate the 3rd International Standard for Hepatitis C virus RNA nucleic acid amplification technology (NAT)‐based assays

A collaborative study was undertaken to evaluate a replacement World Health Organization International Standard for hepatitis C virus (HCV) RNA for nucleic acid amplification technology (NAT)-based assays. The candidate preparations were calibrated in International Units (IUs). Three new candidate preparations were produced from a single bulk containing anti-HCV-negative, genotype 1a HCV RNA-positive plasma. Two samples were lyophilized (coded Sample 2 and Sample 3), whilst a third (Sample 4) contained liquid/frozen material. The samples were distributed together with the 2(nd) International Standard (Sample 1, NIBSC code 96/798) for evaluation by thirty-three laboratories, from fourteen countries. The panel of samples were assayed on four separate occasions. Stability studies were performed for the lyophilized samples by accelerated thermal degradation. Participants returned data from a wide range of commercial and in-house quantitative and qualitative assays. Twenty-five data sets were returned for quantitative assays and fourteen for qualitative assays. Excellent agreement was observed between laboratories and assay methods. The mean relative potencies of Samples 2-4 were 5·19, 5·41 and 5·70 log(10) IU/ml, respectively, when compared against the 2(nd) International Standard. Samples 2 and 3 demonstrated stability of a similar order to the previous standards. Based upon the results of the collaborative study, Sample 2 (code number 06/100) was established as the 3rd International Standard for HCV RNA with an assigned unitage of 5·19 log(10) IU/ml. Each vial contains the equivalent of 0·5 ml of material; each vial contains 4·89 log(10) IU of HCV RNA.

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

BackgroundThe real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories.Principal FindingsHere we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required.ConclusionsThe combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

Suitability of an automated nucleic acid extractor (easyMAG) for use with hepatitis C virus and human immunodeficiency virus type 1 nucleic acid amplification testing

Serological screening assays have greatly reduced, but not eliminated, the risk of transmission of viral infections by transfusion of blood and blood products. In addition, the 1999 regulation of the European Agency for the Evaluation of Medicinal Products requiring all plasma for fractionation to have tested negative for hepatitis C virus (HCV) RNA (CPMP/BWP/390/97, 1998) led many blood transfusion services to introduce nucleic acid amplification technology (NAT) to screen blood donations for HCV, and in some services for human immunodeficiency virus (HIV) and hepatitis B virus (HBV). BioMérieux's second-generation system, the NucliSENS easyMAG, was evaluated as a suitable platform for the automated extraction of nucleic acids for use with the existing SNBTS NAT assays. Two nucleic acid extraction protocols were examined, either lysis on the easyMAG (on board) or a 30-min pre-incubation of the sample with lysis buffer at 37°C (off board). Off board lysis was found to extract nucleic acid more efficiently for both HCV and HIV NAT assays although the improvement was more marked with HIV. The 95% limit of detections (LODs) were 10.11IU/ml (on board) and 7.21IU/ml (off board) for HCV and 55.11IU/ml (on board) and 34.13 (off board) for HIV. Using the more sensitive off board lysis, nucleic acid extraction specificity, robustness and reliability of the easyMAG were examined and over 10,000 Scottish blood donations (in 107 pools of 95 donations) were tested for HCV and HIV in parallel with the existing assay. The results indicate that the easyMAG is a suitable and flexible nucleic acid extraction system, providing high quality nucleic acids and a rapid response alternative to commercial, fully automated, approved blood screening platforms.

Preanalytical stability of HIV‐1 and HCV RNA: impact of storage and plasma separation from cells on blood donation testing by NAT

The aim of this study was to evaluate the optimal preanalytical conditions prior to nucleic acid amplification technology (NAT) for human immunodeficiency virus-1 (HIV-1) or Hepatitis C virus (HCV) RNA in pools of 96 plasma specimens with regard to storage temperature, time and plasma separation in a blood donation environment. Changes in viral nucleic acid concentration of HIV-1 and HCV were observed for 5 days according to the Paul-Ehrlich-Institute's (PEI) guidelines that demand 95%-detection limit of at least 10 000 IU mL(-1) for HIV-1 RNA and 5000 IU mL(-1) for HCV RNA within a single donor blood specimen. Ninety-five per cent detection limits of HIV-1 RNA over 3 days after storage at either 5 or 21 °C were evaluated by using standardised HIV-1 RNA-positive plasma. HCV RNA in whole blood samples proved to be more stable than HIV-1 RNA. Whole blood storage at 21 °C was shown to decrease the detectability of HIV-1 RNA even after only 18 h. Plasma samples once used for NAT at time 18 h did not alter viral stability up to 48 h after donation. Ninety-five per cent detection limits of HIV-1 RNA were securely below 10 000 IU mL(-1) for 24 h after whole blood storage at 5 °C. These results may lead to a discussion around the most suitable preanalytical conditions in blood donation environments. Contrary to the current PEI guidelines that allow storage of whole blood specimens up to 18 h at 21 °C, these results suggest that immediate storage in a 5 °C container after blood donation is more suitable and would permit storage of whole blood up to 24 h prior to the separation of plasma from cells.

Comparison and quality control between domestic and foreign hepatitis virus nucleic acid amplification technology reagents for quantitative and qualitative tests

Hepatitis virus NAT reagents are now widely used clinically. However, the qulity of domestic and foreign NAT reagents varies dramatically. The main reasons for these differences including the manufacture technique, test principle and assay procedure were discussed in this paper and current status of the quality control of the NAT reagents were also described. Finally, it was pointed out that strengthening public supervision and laboratory internal control are very important for the quality improvement of the domestic reagents. Key words: Hepatitis viruses; RNA, viral; DNA, viral; Indicators and reagents; Quality control

False-Positive Transcription-Mediated Amplification Assay Detection of West Nile Virus in Blood from a Patient with Viremia Caused by an Usutu Virus Infection

Detection of West Nile virus (WNV) by nucleic acid amplification technology (NAAT) is used widely to screen blood and organ donations in areas where WNV is endemic. We report a false-positive result of a WNV transcription-mediated amplification assay (TMA) in a patient with viremia that was caused by Usutu virus, a mosquito-borne flavivirus.

Simplified Automated Image Analysis for Detection and Phenotyping of Mycobacterium tuberculosis on Porous Supports by Monitoring Growing Microcolonies

BackgroundEven with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings.MethodsHere we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies “computer vision” and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures.SignificanceMonitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation.

The implementation of nucleic acid amplification technology testing for living tissue donors

There is a significant requirement within the United Kingdom for tissue grafts from living donors. To ensure safety, blood samples from these donors are tested for pathogens at donation, and at 180 days post donation. Nucleic acid amplification technology (NAT) permits more sensitive detection of pathogens in blood samples than serum antigen testing. NAT testing can be applied to samples from living tissue donors to eliminate the need to re-test these donors 180 days post-donation before grafts can be implanted. This has major financial and operational advantages for a tissue bank, and this manuscript describes how NAT testing was assessed and implemented by NHSBT Tissue Services. When compared to traditional serum antigen testing, NAT testing was more cost effective, more convenient for donors and resulted in a greater proportion of donated grafts being made available for transplant.

Risk for Transmission of Pandemic (H1N1) 2009 Virus by Blood Transfusion

To the Editor: Influenza A pandemic (H1N1) 2009 virus emerged in early 2009 in Mexico and has since spread worldwide. In Japan, the first outbreak of the novel influenza was reported in May 2009 (1) and became pandemic in November. Although no cases of transfusion-transmitted influenza have been published, evidence exists of brief viremia before onset of symptoms (2,3). The possibility of transmission of this virus through transfusion of donated blood is of concern. The Japanese Red Cross Blood Centers have intercepted blood products with accompanying postdonation information indicating possible pandemic (H1N1) 2009 infection and attempted to identify the viral genome in those products by using nucleic acid amplification technology (NAT). During June–November 2009, blood samples were collected from plasma and erythrocyte products that had been processed from donations; postdonation information indicated diagnosis of pandemic (H1N1) 2009 infection soon after donation. Viral RNA was extracted from plasma samples and erythrocyte fractions by using a QIAamp Virus Biorobot MDx kit (QIAGEN, Valencia, CA, USA) and a High Pure Viral Nucleic Acid Large Volume kit (Roche Diagnostics, Indianapolis, IN, USA), respectively. RNA samples were subjected to real-time reverse transcription–PCR (RT-PCR) of hemagglutinin (HA) and matrix (M) genes of influenza A by using PRISM 7900 (Applied Biosystems, Foster City, CA, USA). The RT-PCR of HA was specific for pandemic (H1N1) 2009 virus, whereas the RT-PCR of M was designed to detect both pandemic (H1N1) 2009 and seasonal influenza A viruses. The sequences of probes and primers were synthesized according to the protocols developed by the Japanese National Institute of Infectious Diseases (4). Either 200 μL of a plasma sample or 100 µL of packed erythrocytes was used for each test, and the test was performed 2× for each gene in each sample. Before the investigation using donated blood samples, the sensitivity of the NAT system was checked by spiking experiments. Viral particles of pandemic (H1N1) 2009 virus (A/California/04/2009 [H1N1]), donated by the National Institute of Infectious Diseases, were spiked into plasma and erythrocyte samples from healthy volunteers. Viral RNA was detected in the plasma samples spiked with viral particles corresponding to 300 genome equivalents/mL and in the packed erythrocyte samples spiked with viral particles corresponding to 3,000 genome equivalents/mL. NAT was conducted by using 96 plasma and 67 erythrocyte samples obtained from 96 blood donors who had symptoms of influenza within 7 days postdonation. For 20 donors, pandemic (H1N1) 2009 was diagnosed within 1 day postdonation and, for another 20, within 2 days postdonation (Figure). Pandemic (H1N1) 2009 virus was not found in any of the samples tested, but it was consistently detected in the external positive control. These results suggest that the viremia with pandemic (H1N1) 2009 virus, if any, is very low and can be missed by current NAT or that the viremic period is too brief to identify viremia. Although the risk for transmission of pandemic influenza by transfusion seems to be low, further investigation is needed to elucidate this risk. Figure Number of blood donations from persons for whom pandemic (H1N1) 2009 infection was diagnosed postdonation and time between donation and diagnosis, by donor age, Japan.

Collaborative study to establish a replacement World Health Organization International Standard for parvovirus B19 DNA nucleic acid amplification technology (NAT)‐based assays

The aim of the study was to replace the 1(st) World Health Organization International Standard for parvovirus B19 DNA for nucleic acid amplification technique (NAT)-based assays (code 99/800). Two lyophilized preparations (coded 99/800 and 99/802) had been evaluated in the original collaborative study. The present study re-evaluates these two preparations in terms of potency, stability and encapsidation of virus DNA. The 1(st) International Standard (99/800) and 99/802 were re-coded as Samples 1 and 2, respectively. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after storage at 20 degrees C for 7 years. Nuclease treatment was used to investigate the encapsidation of virus DNA. Data were returned from a total of six different quantitative NAT-based assays. The results of the present study confirm those of the original, with no significant differences found in estimated international units (IU)/ml for the 1(st) International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (99/802). Accelerated thermal degradation studies demonstrate that both samples are very stable, with no loss of potency after storage at 20 degrees C for 7 years. Both lyophilized preparations contained the majority of B19V DNA encapsidated in virions. On the basis of the data presented in this collaborative study, Sample 2 (code number 99/802) was established as the 2(nd) International Standard for parvovirus B19 DNA for NAT-based assays with a potency of 10(6) IU/ml (500 000 IU/vial).

Quarantine Plasma: Quo vadis?

Upon the introduction of mandatory nucleic acid amplification technology (NAT) testing in Germany for HCV, quarantining of fresh frozen plasma (FFP) was reduced in 2002 from 6 to 4 months. In 2004 HIV-1 NAT and in 2005 anti-HBc testing were introduced to further reduce the residual transmission risks for transfusion relevant viruses. After testing more than 40 million donations by HCV NAT it became obvious that NAT testing has a very significant impact on viral blood safety by reducing the residual risk by a factor of 10. Only one documented HCV transmission occurred during more than 10 years of NAT testing in Germany, indicating that the remaining risk is marginal. Similar data were obtained for HIV-1. The question arises whether we could discontinue quarantining of FFP or further reduce the quarantining interval for retesting of the donor. This could facilitate logistics and reduce losses as quarantine FFP can be released earlier after donation and at regular donation intervals. Essential parameters for estimating the remaining infectious risks are the minimal infectious dose and replication kinetics of the viruses involved, the detection limits of the NAT tests applied, and the volume of plasma transfused. In essence it can be assumed that discontinuation of quarantining would only marginally increase the residual risk and that the reduction of the quarantine period to only 4 weeks would add an additional benefit to the viral safety of quarantine FFP.

Digital PCR on a SlipChip

This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.