Nucleic Acid Amplification Technology Research Articles

A highly sensitive self-powered sensing method designed on DNA circuit strategy and MoS2 hollow nanorods for detection of thalassemia

Thalassemia is one of the most common monogenic diseases, which seriously affects human growth and development, cardiovascular system, liver, etc. There is currently no effective cure for this disease, making screening for thalassemia particularly important. Herein, a self-powered portable device with high sensitivity and specificity for efficiently screening of low-level thalassemia is developed which is enabled with AuNPs/MoS2@C hollow nanorods and triple nucleic acid amplification technologies. It is noteworthy that AuNPs/MoS2@C electrode shows the advantages of high electrocatalytic activity, fast carrier migration rate and large specific surface area, which can significantly improve the stability and output signal of the platform. Using high-efficiency tetrahedral DNA as the probe, the target CD122 gene associated with thalassemia triggers a catalytic hairpin assembly reaction to achieve CD122 recycling while providing binding sites for subsequent hybridization chain reaction, greatly improving the detection accuracy and sensitivity of the device. A reliable electrochemical/colorimetric dual-mode assay for CD122 is then established, with a linear response range of 0.0001–100 pM for target concentration and open circuit voltage, and the detection limit is 78.7 aM (S/N = 3); a linear range of 0.0001–10000 pM for CD122 level and RGB Blue value, with a detection limit as low as 58.5 aM (S/N = 3). This method achieves ultra-sensitive and accurate detection of CD122, providing a new method for the rapid and accurate screening of thalassemia.

Sensitive detection of cancer biomarker with enzyme-free mediated cascade signal amplification empowered undisturbed dual-mode assay

Cancer is a serious threat to human health, and early identification of cancer biomarkers is crucial for timely treatment. A dual-mode biosensing platform has been developed to identify colon carcinoma suppressor microRNA-199a (miRNA-199a). A biofuel cell is firstly constructed using hybridization chain reaction and catalytic hairpin assembly self-assembly. The anode is a flexible carbon cloth (CP) loaded with glucose oxidase-functionalized molybdenum disulfide nanorods (MoS2 nanorods), and the bio-cathode is made up of double-stranded deoxyribonucleotide chains generated by nucleic acid amplification technology. The linear range of the electrochemical method is 0.1 fM-100 pM, and the limit of detection is 24.1 aM (S/N = 3). The colorimetric method has a linear range of 0.1 fM-10000 pM with detection limit of 34.5 aM (S/N = 3). The combination of MoS2 nanorods, enzyme-free cascade signal amplification technology, and dual-mode detection strategy provides high sensitivity and accuracy, offering a promising method for accurate determination of cancer biomarkers.

How South Africa Used National Cycle Threshold (Ct) Values to Continuously Monitor SARS-CoV-2 Laboratory Test Quality.

The high demand for SARS-CoV-2 tests but limited supply to South African laboratories early in the COVID-19 pandemic resulted in a heterogenous diagnostic footprint of open and closed molecular testing platforms being implemented. Ongoing monitoring of the performance of these multiple and varied systems required novel approaches, especially during the circulation of variants. The National Health Laboratory Service centrally collected cycle threshold (Ct) values from 1,497,669 test results reported from 6 commonly used PCR assays in 36 months, and visually monitored changes in their median Ct within a 28-day centered moving average for each assays' gene targets. This continuous quality monitoring rapidly identified delayed hybridization of RdRp in the Allplex™ SARS-CoV-2 assay due to the Delta (B.1.617.2) variant; S-gene target failure in the TaqPath™ COVID-19 assay due to B.1.1.7 (Alpha) and the B.1.1.529 (Omicron); and recently E-gene delayed hybridization in the Xpert® Xpress SARS-CoV-2 due to XBB.1.5. This near "real-time" monitoring helped inform the need for sequencing and the importance of multiplex molecular nucleic acid amplification technology designs used in diagnostics for patient care. This continuous quality monitoring approach at the granularity of Ct values should be included in ongoing surveillance and with application to other disease use cases that rely on molecular diagnostics.

Tandem CRISPR nucleases-based lateral flow assay for amplification-free miRNA detection via the designed “locked RNA/DNA” as fuels

Currently, available biosensors based on CRISPR/Cas typically depend on coupling with nucleic acid amplification technologies to enhance their sensitivity. However, this approach often involves intricate amplification processes, which could be time-consuming and susceptible to contamination. In addition, signal readouts often require sophisticated and cumbersome equipment, obstructing the applicability of CRISPR/Cas assays in resource-limited regions. Herein, a tandem CRISPR/Cas13a/Cas12a mechanism (tanCRISPR) has been developed via the designed “Locked RNA/DNA” probe as fuels for the trans-cleavage nucleic acid of Cas13a and activated nucleic acid of Cas12a. Meanwhile, a lateral flow assay (LFA) is designed to combine with this tandem CRISPR/Cas13a/Cas12a mechanism (termed tanCRISPR-LFA), realizing the portable monitoring of miRNA-21. The tanCRISPR could realize the limit of detection at pM levels (266 folds lower than Cas13a-based miRNA testing alone) without the resort to target amplification procedures. Furthermore, the miRNA-21 levels of MDA-MB-231 cell extracts are sensed by tanCRISPR-LFA, which is comparable to qRT-PCR. With the virtues of portability, rapidity, sensitivity, and low cost, tanCRISPR-LFA is amenable for CRISPR/Cas-based biosensing and potential applications in the clinical diagnosis of miRNA-associated diseases.

Construction of rolling circle amplification products-based pure nucleic acid nanostructures for biomedical applications.

Nucleic acid nanomaterials with good biocompatibility, biodegradability, and programmability have important applications in biomedical field. Nucleic acid nanomaterials are usually combined with some inorganic nanomaterials to improve their biological stability. However, undefined toxic side effects of composite nanocarriers hamper their application in vivo. As a nanotool capable of avoiding potential biotoxicity, nanostructures composed entirely of DNA oligonucleotides have been rapidly developed in the field of biomedicine in recent years. Rolling circle amplification (RCA) is an isothermal enzymatic nucleic acid amplification technology for large-scale production of periodic DNA/RNA with pre-designed desirable structures and functions. RCA products with different functional parts can be customized by changing the sequence of the circular template, thereby generating complex multifunctional DNA nanostructures, such as DNA nanowire, nanoflower, origami, nanotube, nanoribbon, etc. More importantly, RCA products as nonnicked building blocks can enhance the biostability of DNA nanostructures, especially in vivo. These RCA products-based nucleic acid nanostructures can be used as scaffolds or nanocarriers to interact or load with metal nanoparticles, proteins, lipids, cationic polymers, therapeutic nucleic acids or drugs, etc. This paper reviews the assembly strategies of RCA based DNA nanostructures with different shape and their applications in biosensing, bioimaging and biomedicine. Finally, the development prospects of the nucleic acid nanomaterials in clinical diagnosis and treatment of diseases are described. STATEMENT OF SIGNIFICANCE: As a nanotool capable of avoiding potential biotoxicity, nanostructures composed entirely of DNA oligonucleotides have been rapidly developed in the field of biomedicine in recent years. Rolling circle amplification (RCA) is an isothermal enzymatic nucleic acid amplification technology for large-scale production of periodic DNA/RNA with pre-designed desirable structures and functions. This paper reviews the construction of various shapes of pure nucleic acid nanomaterials based on RCA products and their applications in biosensing, bioimaging and biomedicine. This will promote the development of biocompatible DNA nanovehicles and their further application in living systems, including bioimaging, molecular detection, disease diagnosis and drug delivery, finally producing a significant impact in the field of nanotechnology and nanomedicine.

SARS-CoV-2 Testing Strategies in the Diagnosis and Management of COVID-19 Patients in Low-Income Countries: A Scoping Review.

The accuracy of diagnostic laboratory tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can impact downstream clinical procedures in managing and controlling the outbreak of coronavirus disease 2019 (COVID-19). To assess the effectiveness of laboratory tools for managing COVID-19 patients in low-income countries (LICs), we systematically searched the PubMed, Embase, Scopus and CINHAL databases for reports published between January 2020 and June 2022. We found that 22 of 1303 articles reported the performance of various SARS-CoV-2 detection tools across 10 LICs. These tools were (1) real-time reverse transcriptase polymerase chain reaction (RT-PCR); (2) reverse transcription loop-mediated isothermal amplification (RT-LAMP); (3) rapid diagnostic tests (RDTs); (4) enzyme-linked immunosorbent assay (ELISA); and (5) dot-blot immunoassay. The detection of COVID-19 is largely divided into two main streams-direct virus (antigen) detection and serology (immunoglobulin)-based detection. Point-of-care testing using antigen-based RDTs is preferred in LICs because of cost effectiveness and simplicity in the test procedures. The nucleic acid amplification technology (RT-PCR and RT-LAMP) has the highest diagnostic performance among the available tests, but it is not broadly used in this context due to costs and shortage of facilities/trained staff. The serology-based test method is affected by antibody interferences and varying amounts of SARS-CoV-2 immunoglobulins expressed at different stages of disease onset. We further discuss the effectiveness and shortcomings of each of these tools in the diagnosis and management of COVID-19. Using the LICs as the study model, our findings highlight ways to improve the quality and turnaround time of COVID-19 testing in resource-constrained settings, notably through local/international collaborative efforts to refine the molecular-based or immunoassay-based testing technologies.

Enzyme-Assisted Nucleic Acid Amplification in Molecular Diagnosis: A Review.

Infectious diseases and tumors have become the biggest medical challenges in the 21st century. They are driven by multiple factors such as population growth, aging, climate change, genetic predispositions and more. Nucleic acid amplification technologies (NAATs) are used for rapid and accurate diagnostic testing, providing critical information in order to facilitate better follow-up treatment and prognosis. NAATs are widely used due their high sensitivity, specificity, rapid amplification and detection. It should be noted that different NAATs can be selected according to different environments and research fields; for example, isothermal amplification with a simple operation can be preferred in developing countries or resource-poor areas. In the field of translational medicine, CRISPR has shown great prospects. The core component of NAAT lies in the activity of different enzymes. As the most critical material of nucleic acid amplification, the key role of the enzyme is self-evident, playing the upmost important role in molecular diagnosis. In this review, several common enzymes used in NAATs are compared and described in detail. Furthermore, we summarize both the advances and common issues of NAATs in clinical application.

Tuberculosis Diagnosis Using Isothermal Nucleic Acid Amplification in a Paper-and-Plastic Device.

Nucleic acid (DNA/RNA) amplification technologies are indispensable for applications like disease diagnostics, forensics, epidemiology, evolutionary biology, vaccine development, and therapeutics. While polymerase chain reaction (PCR) has deeply penetrated the abovementioned fields and has been commercially successful, two major common disadvantages are exorbitant costs of associated equipment, which create concerning roadblocks in terms of affordability and accessibility. This work describes development of an inexpensive, portable, easy-to-use and deliverable-to-end-users, nucleic acid amplification technology for infectious disease diagnosis. The device uses loop-mediated isothermal amplification (LAMP) and cell phone-based fluorescence imaging to enable nucleic acid amplification and detection. A regular lab incubator and a custom-made low-cost imaging box are the only two additional equipment required for testing. Material cost for a 12-test zone device was $0.88, and cost of reagents per reaction was $0.43. First successful application of the device was demonstrated for tuberculosis diagnosis with clinical sensitivity of 100% and clinical specificity of 68.75% for testing of 30 clinical patient samples.

Isothermal nucleic acid amplification technology in HIV detection.

Nucleic acid testing for HIV plays an important role in the early diagnosis and monitoring of antiretroviral therapy outcomes in HIV patients and HIV-infected infants. Currently, the main molecular diagnostic methods employed are complex, time-consuming, and expensive to operate in resource-limited areas. Isothermal nucleic acid amplification technology overcomes some of the shortcomings of traditional assays and makes it possible to use point-of-care tests for molecular HIV detection. Here, we summarize and discuss the latest technological advances in isothermal nucleic acid amplification for HIV detection, with the intent of providing guidance for the development of subsequent HIV assays with high sensitivity and specificity.

Molecular Beacons - Loop-Mediated Amplification (MB-LAMP).

High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or single-nucleotide polymorphisms (SNPs). MBs have been adapted for use with the isothermal nucleic acid amplification technology loop-mediated amplification (LAMP) by targeting single-stranded loop sequences under optimized conditions to enable applications such as plant genotyping. LAMP has several benefits over PCR, such as rapid amplification, single-temperature reaction conditions enabling low-cost equipment, and robustness to certain PCR inhibitors. However, and despite the increased number of primers required, the specificity of LAMP is limited, and false positive results can be problematic. In this chapter, design considerations for molecular beacons in LAMP assays are described, as well as a method for MB-LAMP amplification and detection, with an example of gene sequences in genetically modified (GM) maize samples.

325. Molecular Characterization of Antimicrobial Resistance Genes in Neisseria gonorrhoeae in Southeast Michigan

Abstract Background Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG) is a public health crisis. Diagnosis of infection is primarily established using nucleic acid amplification technologies (NAATs) without susceptibility testing; thus rapid identification of resistance determinants is crucial. Whole-genome sequencing (WGS) is a promising alternative to current susceptibility methods. The purpose of this study was to describe AMR genes in a subset of isolates using WGS from Southeast Michigan. Methods Isolates, demographic data, and minimum inhibitory concentrations (MIC) via E-test (ceftriaxone/CRO) and broth microdilution (azithromycin/AZM) were obtained from the Michigan Department of Health and Human Services. Libraries from isolates were generated using QIAseq FX DNA library kit before sequencing on NovaSeq 6000. Reads were trimmed [Trimmomatic] and aligned to the NG genome [Burroughs-Wheeler Aligner] before processing with Samtools suite for variant detection and consensus WGS determination. AMRFinderPlus was used to categorize AMR genes and resistance-associated point mutations. Results Total of 38 isolates were analyzed. The majority were from males (63%) and Blacks (44.7%) living in Detroit City proper (47.3%) with median age 25 (range 13-56), unknown HIV status (55.2%) and sexual orientation (47.3%) (Table 1). Urine (47.3%) was the most common specimen source, followed by cervix/vagina (28.9%). More than a third had prior NG and other sexually transmitted infections. Eight resistance genes were found among the 38 isolates (Figure 1). The penA gene associated with cephalosporin resistance was found in all isolates; however, all isolates were susceptible to CRO (CLSI susceptible breakpoint MIC < 0.25). The mtrR gene associated with macrolide resistance was noted in 86.8% isolates. Among 33 isolates where AZM susceptibility was available, resistance was noted in 9 (27%) (CLSI resistance breakpoint MIC > 1). Conclusion We describe a potential utility for WGS to identify AMR genes in NG. Although numerous β-lactam resistance genes were detected in all isolates, all remained susceptible to CRO. Further research is needed to determine the extent to which genetic diversity affects phenotypic susceptibilities in NG. Disclosures All Authors: No reported disclosures.

Development and characterization of secondary standards for nucleic acid amplification technology (NAAT) assays for detection of hepatitis E virus.

To harmonize assays for detection of HEV RNA, a World Health Organization International Standard (WHO IS) was established. The WHO IS represents the highest order standard for HEV RNA but is limited in quantity. Secondary standards are needed to limit the use of WHO IS and minimize the need to replace it. Establish secondary standards for HEV NAAT assays and to calibrate these against the WHO IS. Stocks of genotype 3 HEV were prepared using both cell lysates and cell culture supernatants to produce non-enveloped and quasi-enveloped virus stocks, respectively. Both stocks were heat-inactivated, diluted in negative human plasma, and lyophilized to produce two candidate secondary standards: HEV-RR (non-enveloped virus) and HEV-RR.1 (quasi-enveloped virus). Both candidate standards were characterized and calibrated against the WHO IS for HEV RNA in an international collaborative study. The collaborative study returned a total of 15 data sets, with different RNA extraction and amplification methods. The estimated mean values relative to the WHO IS (250,000 IU/ml) are 229,000 IU/ml and 355,000 IU/ml for HEV-RR and HEV-RR.1, respectively. We have established two secondary standards for HEV RNA calibrated against the WHO IS. These standards are non-infectious and stable under different storage temperatures.

Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review.

Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.

Robust DNA Cross-Linked Polymeric Lighting-Up Nanogel Facilitates Sensitive Live Cell MicroRNA Imaging.

Accurate and specific imaging of low-abundance intracellular microRNAs (miRNAs) is crucial for monitoring cellular processes and disease diagnosis. Despite the fact that nucleic acid amplification technologies have shown great advantages for the detection of trace targets, their live cell imaging applications remain a major challenge because of the insufficient stability and slow reaction kinetics of the probes in cellular delivery and imaging. Herein, we demonstrate the synthesis of DNA-cross-linked polymeric lighting-up nanogels (DPLNs) through the DNA hairpin-based hybridization chain reaction within nanoscale-confined space for monitoring and imaging live cell miRNA-21 with high sensitivity. Cascaded catalytic hairpin assembly of two hairpin signal probes confined in the DPLNs can be triggered by the target miRNA, causing substantially amplified fluorescence resonance energy transfer signals with accelerated reaction kinetics. Moreover, the DPLNs show low cytotoxicity and highly enhanced nuclease resistance and can be successfully delivered into live cells for imaging low levels of miRNA-21. In addition, the DPLN probes can be readily tuned by specific sequences for monitoring various molecular targets in live cells for important biological and biomedical applications.

Perspectives of physicians and medical assistants on the implementation of NAAT-based point-of-care testing for SARS-CoV-2 in primary care in Germany

IntroductionGiven their high diagnostic accuracy and fast turnaround time, rapid SARS-CoV-2 tests based on nucleic acid amplification technologies (NAAT) have great potential to expand access to testing and decrease delays in diagnosis of COVID-19. ObjectivesThe aim of this study was to investigate feasibility, acceptance, organizational consequences and other implementation aspects of the use of a NAAT-based SARS-CoV-2 rapid test (ID NOW™ COVID-19, Abbott Diagnostics) for symptomatic primary care patients with a suspected SARS-CoV-2 infection. MethodsCross-sectional survey among primary care physicians and medical assistants from Thuringia (Germany) during the third wave of the COVID-19 pandemic in March and April 2021. The health care providers surveyed had previously used the NAAT-based SARS-CoV-2 rapid test as part of a pilot study. ResultsEleven physicians (ten general practitioners and one paediatrician) and 22 medical assistants from Thuringia (Germany) participated in the written survey. Four physicians were additionally interviewed. The majority of the surveyed health care providers rated user-friendliness, integration into practice routine, impact on communication with patients and technical reliability of the NAAT-based SARS-CoV-2 rapid test as (very) positive. Greater workload and the costs for measuring devices were identified as disadvantages compared to PCR laboratory tests. Four out of ten physicians rated the lower sample turnover as unfavourable. ConclusionOur survey shows that NAAT-based point-of-care SARS-CoV-2 testing gained widespread acceptance among physicians and medical assistants, positively influences workflows, can improve patient communication and could therefore be successfully implemented into routine primary care.

Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV)

BackgroundSwine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV. ResultsThe detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results.ConclusionsThese results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.

The Microbial Etiology of Community-Acquired Pneumonia in Adults: from Classical Bacteriology to Host Transcriptional Signatures.

All modern advances notwithstanding, pneumonia remains a common infection with substantial morbidity and mortality. Understanding of the etiology of pneumonia continues to evolve as new techniques enable identification of already known organisms and as new organisms emerge. We now review the etiology of pneumonia (at present often called "community-acquired pneumonia") beginning with classic bacteriologic techniques, which identified Streptococcus pneumoniae as the overwhelmingly common cause, to more modern bacteriologic studies, which emphasize Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Enterobacteriaceae, Pseudomonas, and normal respiratory flora. Urine antigen detection is useful in identifying Legionella and pneumococcus. The low yield of bacteria in recent studies is due to the failure to obtain valid sputum samples before antibiotics are administered. The use of high-quality sputum specimens enables identification of recognized ("typical") bacterial pathogens as well as a role for commensal bacteria ("normal respiratory flora"). Nucleic acid amplification technology for viruses has revolutionized diagnosis, showing the importance of viral pneumonia leading to hospitalization with or without coinfecting bacterial organisms. Quantitative PCR study of sputum is in its early stages of application, but regular detection of high counts of bacterial DNA from organisms that are not seen on Gram stain or grown in quantitative culture presents a therapeutic dilemma. This finding may reflect the host microbiome of the respiratory tract, in which case treatment may not need to be given for them. Finally, host transcriptional signatures might enable clinicians to distinguish between viral and bacterial pneumonia, an important practical consideration.

Characterizing the Impact of Primer-Template Mismatches on Recombinase Polymerase Amplification

Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3'-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping.

Follow-Up of Plasma Donors after Quarantine Storage: Results of a 5-Year Survey in Germany

Background and Objectives: In 1993, a quarantine storage of 6 months was introduced for plasma for transfusion and was reduced to 4 months in 2003, owing to the improvements of screening assays used in German blood establishments. The presented survey analyses the value of quarantine storage under the current screening conditions. Materials and Methods: From 2015 to 2019, we collected data on the total amount of released quarantine plasma as well as on the number of quarantine plasma not released due to a reactive screening test of a follow-up donation. Results: Responding establishments covered 84% of plasma units released within the sampled period. In 3,583,913 (99.98%) of the total 3,584,664 test pairs, all screening assays revealed a negative result, leading to plasma release from quarantine storage. In 442 out of the residual 751 cases, confirmed positive results for human immunodeficiency virus (24), hepatitis C virus (22), or hepatitis B virus (396) were obtained in the follow-up donations. Of them, 372 revealed negative ID-NAT results in their retain samples confirmed by using highly sensitive individual donor nucleic acid amplification technology. In 70 cases, no testing of retain samples was performed as plasma was released for fractionation. The maximum theoretical risk for an undetected human immunodeficiency virus, hepatitis C or B virus infection was less than 0.0001%. Conclusion: No positive donation were found under the current screening regime and the quarantine storage during the 5-year survey period. In view of the current type and sensitivity of screening tests in German blood establishments, the results allow a reassessment of the value of quarantine storage of plasma regarding duration and release modalities. Due to the more sensitive donor screening, shorter quarantine periods as well as dispensing quarantine storage can be discussed. A reduction in the safety standard of plasma transfusions need not be feared, and the availability of plasma for transfusions could be facilitated.

Research progress in rolling circle amplification in biosensing and cell imaging

<p indent="0mm">Rolling circle amplification (RCA) is an isothermal nucleic acid amplification technology that performs massive amplification of rolling circle replication by hybridizing with target nucleic acid DNA, which has the advantages of simplicity, rapidity and high amplification efficiency. RCA technology has been widely used in the field of biological analysis, especially in the field of sensor detection and cell imaging analysis of nucleic acid, protein, exosome, virus and other disease biomarkers. In this paper, the basic principles and classification of RCA technology are introduced, the research progress of RCA technology in the field of biosensing and cell imaging in recent years is systematically reviewed, and the shortcomings and improvement strategies of RCA technology are proposed.