Multiple Displacement Amplification Research Articles

Development and clinical evaluation of a real-time multiple cross displacement amplification assay for rapid and sensitive detection of Mycobacterium tuberculosis

Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the Mycobacterium tuberculosis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 minutes) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 hour. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

PGT-M for spinocerebellar ataxia type 1: development of a STR panel and a report of two clinical cases.

To present the developed preimplantation genetic testing (PGT) for spinocerebellar ataxia type 1 (SCA1) and the outcomes of IVF with PGT. PGT was performed for two unrelated couples from the Republic of Sakha (Yakutia) with the risk of SCA1 in one spouse. We have developed a system for PGT of a monogenic disease (PGT-M) for SCA1, which includes the analysis of a panel of 11 polymorphic STR markers linked to the ATXN1 gene and a pathogenic variant of the ATXN1 gene using nested PCR and fragment analysis. IVF/ICSI programs were performed according to standard protocols. Multiple displacement amplification (MDA) was used for whole genome amplification (WGA) and array comparative genomic hybridization (aCGH) for aneuploidy testing (PGT-A). Eight STRs were informative for the first couple and ten for the second. Similarity of the haplotypes carrying pathogenic variants of the ATXN1 gene was noted. In the first case, during IVF/ICSI-PGT, three embryos reached the blastocyst stage and were biopsied. One embryo was diagnosed as normal by maternal STR haplotype and the ATXN1 allele. PGT-A revealed euploidy. The embryo transfer resulted in a singleton pregnancy, and a healthy boy was born. Postnatal diagnosis confirmed normal ATXN1. In the second case, two blastocysts were biopsied. Both were diagnosed as normal by PGT-M, but PGT-A revealed aneuploidy. Birth of a healthy child after PGT for SCA1 was the first case of successful preimplantation prevention of SCA1 for the Yakut couple and the first case of successful PGT for SCA1 in Russia.

Abstract 322: Ultraplex genome and exome sequencing of rare single cells

Abstract Single-cell genome analysis enables researchers to gain novel insights into tumor heterogeneity. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging since the amount of genomic DNA present in a single cell is limited. PCR-based amplification methods normally have high error rates, low coverage uniformity, and extensive allelic drop-outs making interpretation of SNVs and CNVs challenging.Here we introduce a new whole-genome amplification method which utilizes an optimized isothermal DNA amplification reaction with a Hi-fidelity, error correcting polymerase and downstream library preparation protocol to allow pooling of amplified DNA. The key DNA amplification steps use multiple displacement amplification (MDA) technology to amplify gDNA directly from single cells, nuclei or previously isolated genomic DNA. During the MDA reaction, sample-barcodes are incorporated into the newly synthetized DNA, allowing the amplified DNA to be pooled and processed in to one ultraplex library. This method saves library preparation time, reduces DNA library costs and limits plastic consumable usage.Our current workflow allows 24 samples to be multiplexed in one library and 24 libraries to be produced for a total of 576 samples per sequencing flow cell. This method can be used to investigate chromosomal instability with low pass sequencing or SNV and CNV analysis following whole genome, exome-capture workflows. Citation Format: Ioanna Andreou, Samuel Rulli, Katja Heitz, Silke Huebner, Christin Meerschiff, Eric Lader. Ultraplex genome and exome sequencing of rare single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 322.

Appropriate whole genome amplification and pathogenic loci detection can improve the accuracy of preimplantation genetic diagnosis for deletional α-thalassemia.

To improve the accuracy of preimplantation genetic testing (PGT) in deletional α-thalassemia patients. Article. fifty-two deletional α-thalassemia couples. Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) methods for PGT for deletional α-thalassemia. Blastocyst biopsy samples (n = 253) were obtained from 52 deletional α-thalassemia couples. The results of the comparison of experimental data between groups MALBAC and MDA are as follows: (i) The average allele drop-out (ADO) rate, MALBAC vs. MDA = 2.27% ± 3.57% vs. 0.97% ± 1.4%, P=0.451); (ii) WGA success rate, MALBAC vs. MDA = 98.61% vs. 98.89%, P=0.851; (iii) SNP haplotype success rate, MALBAC vs. MDA = 94.44% vs. 96.68%, P=0.409; (iv) The result of SNP haplotype analysis is consistent with that of Gap-PCR/Sanger sequencing results, MALBAC vs. MDA = 36(36/72, 50%) vs. 151(151/181, 83.43%), P=0; (v) Valid SNP loci, MALBAC vs. MDA = 30 ± 9 vs. 34 ± 10, P=0.02; (vi) The mean CV values, MALBAC vs. MDA = 0.12 ± 0.263 vs. 0.09 ± 0.40, P=0.916; (vii) The average number of raw reads, MALBAC vs. MDA =3244259 ± 999124 vs. 3713146 ± 1028721, P=0; (viii) The coverage of genome (%), MALBAC vs. MDA = 5.02 ± 1.09 vs. 5.55 ± 1.49, P=0.008. Our findings indicate that MDA is superior to MALBAC for PGT of deletional α-thalassemia. Furthermore, SNP haplotype analysis combined with PCR loci detection can improve the accuracy and detection rate of deletional α-thalassemia.

Biosensor-based multiple cross displacement amplification platform for visual and rapid identification of hepatitis C virus.

Hepatitis C remains a global health problem, especially in poverty-stricken areas. A rapid and sensitive point-of-care (POC) diagnostic tool is critical for the early detection and timely treatment of hepatitis C virus (HCV) infection. Here, for the first time, we reported a novel molecular diagnostic assay, termed reverse transcription multiple cross displacement amplification integrated with a gold-nanoparticle-based lateral flow biosensor (RT-MCDA-AuNPs-LFB), which was developed for rapid, sensitive, specific, and visual identification of HCV. HCV-RT-MCDA induced rapid isothermal amplification through a specific primer set targeting the 5'untranslated region gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a that are prevalent in China. The optimal reaction temperature and time for RT-MCDA-AuNPs-LFB were 68°C and 25 min, respectively. The limit of detection of the assay was 10 copies per test, and the specificity was 100% for the experimental strains. The whole detection procedure, including crude nucleic acid isolation (~5 min), RT-MCDA (68°C, 25 min), and visual AuNPs-LFB result confirmation (less than 2 min), was performed within 35 min. The preliminary results indicated that the HCV-RT-MCDA-AuNPs-LFB assay could be a valuable tool for sensitive, specific, visual, cost-saving, and rapid detection of HCV and has potential as a POC diagnostic platform for field screening and early clinical detection of HCV infection.

Development of a multiple cross displacement amplification combined with nanoparticles-based biosensor assay for rapid and sensitive detection of Streptococcus pyogenes

BackgroundS. pyogenes, is a primary pathogen that leads to pharyngitis and can also trigger severe conditions like necrotizing fasciitis and streptococcal toxic shock syndrome (STSS), often resulting in high mortality rates. Therefore, prompt identification and appropriate treatment of S. pyogenes infections are crucial in preventing the worsening of symptoms and alleviating the disease's impact. ResultsIn this study, a newly developed technique called multiple cross displacement amplification (MCDA) was employed to detect S. pyogenes,specifically targeting the speB gene, at a temperature of 63°C within 30 min. Then, an easily portable and user-friendly nanoparticles-based lateral flow biosensor (LFB) assay was introduced for the rapid analysis of MCDA products in just 2 min. The results indicated that the LFB offers greater objectivity compared to Malachite Green and is simpler than electrophoresis. The MCDA-LFB assay boasts a low detection limit of 200 fg and exhibits no cross-reaction with non-S. pyogenes strains. Among 230 clinical swab throat samples, the MCDA-LFB method identified 27 specimens as positive, demonstrating higher sensitivity compared to 23 samples detected positive by qPCR assay and 18 samples by culture. The only equipment needed for this assay is a portable dry block heater. Moreover, each MCDA-LFB test is cost-effective, priced at approximately $US 5.5.ConclusionThe MCDA-LFB assay emerges as a straightforward, specific, sensitive, portable, and user-friendly method for the rapid diagnosis of S. pyogenes in clinical samples.

A microfluidics-free centrifugally-driven nano-liquid pipetting system (CNPS) for single-cell whole genome amplification

We developed a microfluidics-free centrifugally-driven nano-liquid pipetting system (CNPS) for single-cell whole genome amplification. The capillary tube and the collection vessel (PCR tube or 96-well PCR plate), both of which are easily accessible, make up the basic components of the system. Unlike microfluidic chips that need a clean room and special skills to fabricate, the CNPS can be assembled in the typical biology laboratory without any special training. Centrifugation is used to directly pipette the nanoliter-volume liquid into the collection tube, which eliminates the requirement for extra equipment. The CNPS, which is compatible with the standard handheld pipette, allows for a smooth transition between the micro and macro worlds, thereby overcoming the problem of retrieving nanoliter-volume products. We further demonstrated the feasibility of the CNPS for nanoliter-volume single-cell multiple displacement amplification. Based on the sequencing results, the mapped reads cover on average 95.23%, 95.15%, and 80.94% for Saccharomyces cerevisiae, Escherichia coli, and Staphylococcus epidermidis, respectively. In sum, the CNPS has the potential to become an alternative powerful platform for single-cell genome sequencing with the ability to manipulate ultra-small volumes, as well as to enhance precision and sophistication by significantly minimizing sample losses and reducing amplification bias.

Evaluation of multiple displacement amplification for metagenomic analysis of low biomass samples.

Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for high-throughput sequencing. Although amplification bias and nonspecific amplification have been reported from MDA-amplified samples, the impact of MDA on metagenomic datasets is not well understood. We compared three MDA methods (i.e. bulk MDA, emulsion MDA, and primase MDA) for metagenomic analysis of two DNA template concentrations (approx. 1 and 100pg) derived from a microbial community standard "mock community" and two low biomass environmental samples (i.e. borehole fluid and groundwater). We assessed the impact of MDA on metagenome-based community composition, assembly quality, functional profiles, and binning. We found amplification bias against high GC content genomes but relatively low nonspecific amplification such as chimeras, artifacts, or contamination for all MDA methods. We observed MDA-associated representational bias for microbial community profiles, especially for low-input DNA and with the primase MDA method. Nevertheless, similar taxa were represented in MDA-amplified libraries to those of unamplified samples. The MDA libraries were highly fragmented, but similar functional profiles to the unamplified libraries were obtained for bulk MDA and emulsion MDA at higher DNA input and across these MDA libraries for the groundwater sample. Medium to low-quality bins were possible for the high input bulk MDA metagenomes for the most simple microbial communities, borehole fluid, and mock community. Although MDA-based amplification should be avoided, it can still reveal meaningful taxonomic and functional information from samples with extremely low DNA concentration where direct metagenomics is otherwise impossible.

Multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor for rapid and sensitive detection of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a highly dangerous virus that is globally prevalent and closely linked to the development of nasopharyngeal cancer (NPC). Plasma EBV DNA analysis is an effective strategy for early detection, prognostication and monitoring of treatment response of NPC. Here, we present a novel molecular diagnostic technique termed EBV-MCDA-LFB, which integrates multiple cross displacement amplification (MCDA) with nanoparticle-based lateral flow (LFB) to enable simple, rapid and specific detection of EBV. In the EBV-MCDA-LFB system, a set of 10 primers was designed for rapidly amplifying the highly conserved tandem repeat BamHI-W region of the EBV genome. Subsequently, the LFB facilitate direct assay reading, eliminating the use of extra instruments and reagents. The outcomes showed that the 65°C within 40 minutes was the optimal reaction setting for the EBV-MCDA system. The sensitivity of EBV-MCDA-LFB assay reached 7 copies per reaction when using EBV recombinant plasmid, and it showed 100% specificity without any cross-reactivity with other pathogens. The feasibility of the EBV-MCDA-LFB method for EBV detection was successfully validated by 49 clinical plasma samples. The complete detection process, consisting of rapid template extraction (15 minutes), MCDA reaction (65°C for 40 minutes), and LFB result reading (2 minutes), can be finalized within a 60-minutes duration. EBV-MCDA-LFB assay designed here is a fast, extremely sensitive and specific technique for detecting EBV in field and at the point-of-care (PoC), which is especially beneficial for countries and regions with a high prevalence of the disease and limited economic resources.

Profiling DNA Cargos in Single Extracellular Vesicles via Hydrogel-Based Droplet Digital Multiple Displacement Amplification.

Due to the substantial heterogeneity among extracellular vesicle (EV) subpopulations, single-EV analysis has the potential to elucidate the mechanisms behind EV biogenesis and shed light on the myriad functions, leading to the development of novel diagnostics and therapeutics. While many studies have been devoted to reveal between-EV variations in surface proteins and RNAs, DNA cargos (EV-DNA) have received little attention. Here, we report a hydrogel-based droplet digital multiple displacement amplification approach for the comprehensive analysis of EV-DNA at the single-EV level. Single EVs are dispersed in thousands of hydrogel droplets and lysed for DNA amplification and identification. The droplet microfluidics strategy empowers the assay with single-molecule sensitivity and capability for absolute quantification of DNA-containing EVs. In particular, our findings indicate that 5-40% EVs are associated with DNA, depending on the cell of origin. Large EVs exhibit a higher proportion of DNA-containing EVs and a more substantial presence of intraluminal DNA, compared to small EVs. These DNA-containing EVs carry multiple DNA fragments on average. Furthermore, both double-stranded DNA and single-stranded DNA were able to be detected at the single-EV level. Utilizing this method, the abundance, distribution, and biophysical properties of EV-DNA in various EV populations are evaluated. The DNA level within EVs provides insight into the status of the originating cells and offers valuable information on the outcomes of anticancer treatments. The utilization of single-EV analysis for EV-DNA holds significant promise for early cancer detection and treatment response monitoring.

Preimplantation Genetic Testing for Single Gene Conditions and Aneuploidy

It is now over 30 years since the first pregnancies and live births following IVF, embryo biopsy and preimplantation genetic testing (PGT) were reported in 1990. Since then, methods for genetic analysis at the single-cell level have evolved alongside the rapid advances in human genetics and sequencing of the human genome. With the development of methods for whole genome amplification, such as multiple displacement amplification (MDA), genome-wide analysis is now possible. This has led to the development of microarray or next generation sequencing (NGS) based methods for genome-wide haplotyping of parental chromosomes in embryo samples and enabled universal linkage-based testing for monogenic diseases combined with molecular cytogenetic analysis of chromosome abnormalities. These comprehensive methods have also recently been exploited to calculate polygenic risk scores for common multifactorial conditions, such as diabetes, cardiovascular disease, and cancer. Although controversial, polygenic risk scoring may have a role for reducing residual risk of breast cancer, for example, in combination with PGT for pathogenic variants. Finally, there is now evidence that cell-free DNA in spent culture medium at the blastocyst stage may originate from nuclear DNA shedding from the trophectoderm. In the future, therefore, it may be possible to use this cell-free DNA for genome-wide analysis and comprehensive PGT to avoid embryo biopsy.

Taxonomic Classification of Geminiviruses Based on Pairwise Sequence Comparisons.

Geminiviridae is the largest and one of the most diverse families of plant viruses, comprising 14 genera demarcated based on host range, type of insect vector, and phylogenetic relationships. The use of unbiased, whole-genome multiple displacement amplification techniques coupled with high-throughput sequencing has greatly expanded our knowledge of geminivirus diversity over the last two decades. As a result, a large number of new species have been described in recent years. Species demarcation criteria in the family are entirely based on sequence comparisons, but the specific cutoff values vary for each genus. The purpose of this chapter is to provide a step-by-step pipeline to classify new species in the family Geminiviridae.

Genomic insights into the adaptation of Synechococcus to the coastal environment on Xiamen.

Synechococcus are widely distributed in the global ocean, from the pelagic zone to coastal waters. However, little is known about Synechococcus in coastal seawater due to limitations in isolation and culture conditions. In this study, a combination of metagenomic sequencing technology, flow cytometry sorting, and multiple displacement amplification was used to investigate Synechococcus in the coastal seawater of Xiamen Island. The results revealed 18 clades of Synechococcus and diverse metabolic genes that appear to contribute to their adaptation to the coastal environment. Intriguingly, some metabolic genes related to the metabolism of carbohydrates, energy, nucleotides, and amino acids were found in 89 prophage regions that were detected in 16,258 Synechococcus sequences. The detected metabolic genes can enable prophages to contribute to the adaptation of Synechococcus hosts to the coastal marine environment. The detection of prophages also suggests that the cyanophages have infected Synechococcus. On the other hand, the identification of 773 genes associated with antiviral defense systems indicates that Synechococcus in Xiamen have evolved genetic traits in response to cyanophage infection. Studying the community diversity and functional genes of Synechococcus provides insights into their role in environmental adaptation and marine ecosystems.

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3)

BackgroundIdentification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function.ResultsWe expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3′-5′ exonuclease activity, but does not have 5′-3′ exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5′-3′ polymerase activity and can amplify DNA at 15–35 °C and a pH range of 5.5–9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3′-5′ exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10–6).ConclusionsA novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.

A modified multiple cross displacement amplification linked with a gold nanoparticle biosensor for the detection of Epstein-Barr virus in clinical applications.

Epstein-Barr virus (EBV), a double-stranded DNA virus belonging to the family Herpesviridae, infects more than 95% of healthy adults by attacking the host immune system. Here, a novel detection protocol, utilizing the modified multiple cross displacement amplification (MCDA) technique combined with a gold nanoparticles-based lateral flow biosensors (AuNPs-LFB), was devised and developed to detect EBV infection (termed EBV-MCDA-LFB assay). Ten MCDA primers targeting the EBNA-LP gene were designed, including CP1* primers modified with 6-carboxyfluorescein (FAM) and D1* primers modified with biotin. Then, nucleic acid templates extracted from various pathogens and whole blood samples were used to optimize and evaluate the EBV-MCDA-LFB assay. As a result, the lowest concentration of EBNA-plasmids, which can be detected by MCDA-LFB assay with an optimal reaction condition of 67°C for 30 min, was 10 copies/reaction. Here, the MCDA-LFB assay can detect all EBV pathogens used in the study, and no cross-reactions with non-EBV organisms were observed. Meanwhile, the entire detection workflow of the EBV-MCDA-LFB assay for whole blood samples, including DNA template preparation (25 min), EBV-MCDA amplification (30 min), and AuNPs-LFB-mediated validation (2-5 min), can be completed within 1 h. Taken together, the EBV-MCDA-LFB assay established in the current study is a rapid, simplified, sensitive, specific, and easy-to-obtain technique that can be used as a screening or diagnostic tool for EBV infection in clinical applications, especially in resource-poor regions.

Highly sensitive and rapid determination of Mycobacterium leprae based on real-time multiple cross displacement amplification

BackgroundMycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed.ResultsIn this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor.ConclusionThe E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.

Biosensor-Based Multiple Cross Displacement Amplification for the Rapid Detection of Mycobacterium leprae.

Leprosy is an ancient disease caused by Mycobacterium leprae (ML) that remains a public health problem in poverty-stricken areas worldwide. Although many ML detection techniques have been used, a rapid and sensitive tool is essential for the early detection and treatment of leprosy. Herein, we developed a rapid ML detection technique by combining multiple cross displacement amplification (MCDA) with a nanoparticle-based lateral flow biosensor (LFB), termed ML-MCDA-LFB. MCDA induced a rapid isothermal reaction using specific primers targeting the RLEP gene, and the LFB enabled instant visual amplicon detection. The pure genomic DNA of ML and nucleic acids from various pathogens were employed to evaluate and optimize the ML-MCDA-LFB assay. The optimal conditions for ML-MCDA-LFB were 68 °C and 35 min, respectively. The limit of detection for pure ML genomic DNA was 150 fg per vessel, and the specificity of detection was 100% for the experimental strains. Additionally, the entire detection process could be performed within 40 min, including the isothermal amplification (35 min) and result confirmation (1-2 min). Hence, the ML-MCDA-LFB assay was shown to be a rapid, sensitive, and visual method for detecting ML and could be used as a potential tool for early clinical diagnosis and field screening of leprosy.

Conventional IVF is feasible in preimplantation genetic testing for aneuploidy.

To investigate the feasibility of the application of conventional in vitro fertilization (cIVF) for couples undergoing preimplantation genetic testing for aneuploidies (PGT-A) with non-male factor infertility. To evaluate the efficiency of sperm whole-genome amplification (WGA), spermatozoa were subjected to three WGA protocols: Picoplex, ChromInst, and multiple displacement amplification (MDA). In the clinical studies, 641 couples who underwent PGT-A treatment for frozen embryos between January 2016 and December 2021 were included to retrospectively compare the chromosomal and clinical outcomes of cIVF and intracytoplasmic sperm injection (ICSI). Twenty-six couples were prospectively recruited for cIVF and PGT-A treatment between April 2021 and April 2022; parental contamination was analyzed in biopsied samples; and 12 aneuploid embryos were donated to validate the PGT-A results. Sperm DNA failed to amplify under Picoplex and ChromInst conditions but could be amplified using MDA. In frozen PGT-A cycles, no significant differences in the average rates of euploid, mosaic, and aneuploid embryos per cycle between the cIVF-PGT-A and ICSI-PGT-A groups were observed. The results of the prospective study that recruited couples for cIVF-PGT-A treatment showed no paternal contamination and one case of maternal contamination in 150 biopsied trophectoderm samples. Among the 12 donated embryos with whole-chromosome aneuploidy, 11 (91.7%) presented uniform chromosomal aberrations, which were in agreement with the original biopsy results. Under the Picoplex and ChromInst WGA protocols, the risk of parental contamination in the cIVF-PGT-A cycles was low. Therefore, applying cIVF to couples with non-male factor infertility who are undergoing PGT-A is feasible.