Genomic DNA Template Research Articles

Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR. Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay. We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits. We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

Recombinase-aid amplification combined with lateral flow detection assay for sex identification of the great white pelican (Pelecanus onocrotalus)

Sex identification in avian species is essential for biodiversity conservation and ecological studies. However, the sex of nearly half of the birds could not be identified based on their external appearance. It is difficult to visually identify sex to monitor the ecology and conservation of wild populations. In this study, we designed primer pairs for large white pelican using recombinase-based isothermal amplification combined with a lateral flow dipstick (RAA-LFD) assay for chromo-helicase-DNA binding protein (CHD) genes mapped to W chromosomes and an ultra-conserved element (UCE) located on chromosome 6, respectively. Our result showed that the raaW4-RAA-LFD can detect up to 0.1 ng of genomic DNA (gDNA) templates of female pelicans in 30 min at 39 ℃ and accurately distinguish female from male without any cross reactivity. RaaUCE2-RAA-LFD can amplify both male and female pelicans with a detection limit of 25 pg. To further evaluate the assay, 15 white pelicans of unknown sex were tested using the RAA-LFD assay and conventional polymerase chain reaction (PCR). The results of the raaW4-RAA-LFD assay were consistent with those of the conventional PCR. The developed RAA-LFD assay is equipped with field-deployable instruments and offers a field platform for rapid and reliable sex identification in pelicans.

Polymorphism Identification in the Coding Sequences (ORFs) of the Porcine Pregnancy-Associated Glycoprotein 2-like Gene Subfamily in Pigs.

Pregnancy-associated glycoproteins (PAGs) are a polygenic family with many scattered genes and pseudogenes resulting from the duplication or fusion of a pseudogene with expression beginning in the trophoblast during the peri-implantation period and continuing in the trophectoderm. In this study, single-nucleotide polymorphism (SNP) and insertion/deletion (InDels) in the open reading frame (nine exons) of crossbreed pigs are reported for the first time. Novel SNPs/InDels were researched using genomic DNA templates isolated from the leukocytes of crossbreed pigs (N = 25), which were amplified, gel-out-purified, and sequenced. Sixteen SNPs and one InDel (g.6961_6966 Ins TGCCAA) were identified in the crossbreed pigs. In silico analysis revealed that among 16 SNPs, only 10 SNPs cause amino acid (aa) substitutions, and InDel codes asparagine (N298) and alanine (A299). The results provide a novel broad-based database (main pattern) that will be critical for future research into the possible correlations between the SNP genotypes of the pPAG2-L subfamily in pigs of various breeds whose reproductive traits are known.

Enhancing drought tolerance in Malva parviflora plants through metabolic and genetic modulation using Beauveria bassiana inoculation

BackgroundEnhancing crops’ drought resilience is necessary to maintain productivity levels. Plants interact synergistically with microorganisms like Beauveria bassiana to improve drought tolerance. Therefore, the current study investigates the effects of biopriming with B. bassiana on drought tolerance in Malva parviflora plants grown under regular irrigation (90% water holding capacity (WHC)), mild (60% WHC), and severe drought stress (30% WHC).ResultsThe results showed that drought stress reduced the growth and physiological attributes of M. parviflora. However, those bioprimed with B. bassiana showed higher drought tolerance and enhanced growth, physiological, and biochemical parameters: drought stress enriched malondialdehyde and H2O2 contents. Conversely, exposure to B. bassiana reduced stress markers and significantly increased proline and ascorbic acid content under severe drought stress; it enhanced gibberellic acid and reduced ethylene. Bioprimed M. parviflora, under drought conditions, improved antioxidant enzymatic activity and the plant’s nutritional status. Besides, ten Inter-Simple Sequence Repeat primers detected a 25% genetic variation between treatments. Genomic DNA template stability (GTS) decreased slightly and was more noticeable in response to drought stress; however, for drought-stressed plants, biopriming with B. bassiana retained the GTS.ConclusionUnder drought conditions, biopriming with B. bassiana enhanced Malva’s growth and nutritional value. This could attenuate photosynthetic alterations, up-regulate secondary metabolites, activate the antioxidant system, and maintain genome integrity.

Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates

One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate.

Rapid detection of FadA in Fusobacterium nucleatum using the quantitative LAMP colorimetric phenol red method in stool samples from colorectal cancer patients

The study aimed to develop a quantitative colorimetric loop-mediated isothermal amplification technique using the phenol red indicator (QLAMP-PhR) for detecting Fusobacterium nucleatum (Fn) levels in colorectal cancer (CRC) patients and healthy individuals. QLAMP-PhR assays were conducted on 251 stool samples specific for the Fn FadA gene. Six primers were synthesized and utilized with master mix reagents, and a phenol red indicator was employed to enhance the QLAMP-PhR technique. A standard quantitative analysis curve was generated using a logarithmic function (absorbance vs. concentration) by serially diluting the copy number of genomic DNA templates (Fn ATCC25586). The CRC group exhibited a significantly higher abundance of Fn compared to the healthy control group (P < 0.001). These findings suggest that the QLAMP-PhR technique effectively identifies Fn specifically by its gene for the key virulence factor FadA. Additionally, ideas for developing a real-time QLAMP-PhR test were presented. Compared to the traditional polymerase chain reaction (PCR) technique, QLAMP-PhR offers several advantages including rapidity, simplicity, specificity, sensitivity, and cost-effectiveness method that can quantitatively screen for Fn presence in normal populations. The QLAMP-PhR method represents a sensitive and specific amplification assay for the rapid detection of the Fn pathogen. To the best of our knowledge, this study is the first to report the application of QLAMP-PhR for detecting FadA in Fn.

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 min) 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 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield.

Phage therapy to treat life-threatening drug-resistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (infC) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

Rapid, ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform.

Mycoplasma pneumoniae (MP) is an important causative agent of morbidity and mortality among all age groups, especially among patients of extreme ages. Improved and readily available tests for accurate, sensitive and rapid diagnosis of MP infection is sorely needed. Here, we developed a CRISPR-Cas12b-based detection platform on the basis of recombinase polymerase amplification (RPA) for rapid, simple, and accurate diagnosis of MP infection, named MP-RPA-CRISPR. The RPA was employed for amplifying the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP strains at the optimal reaction temperature 37°C. The resulting amplicons were decoded by the CRISPR-Cas12b-based detection platform, which was interpreted by real-time PCR system and by naked eye under blue light. The MP-RPA-CRISPR can detected down to 5 fg of genomic DNA templates of MP strains and accurately distinguish MP strains from non-MP strains without any cross-reactivity. A total of 96 bronchoalveolar lavage fluid (BALF)samples collected from patients suspected of respiratory infection were used to evaluate the clinical performance of the MP-RPA-CRISPR assay. As a result, our assay accurately diagnosed 45 MP-infected samples and 51 non-MP infected sample, and the results obtained from MP-RPA-CRISPR were consistent with microfluidic chip technology. In conclusion, our MP-RPA-CRISPR assay is a simple, rapid, portable and highly sensitive method to diagnose MP infection, which can be used as a promising tool in a variety of settings including clinical, field, and resource-limited aeras.

Reverse transcription-quantitative PCR (RT-qPCR) without the need for prior removal of DNA

The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA’s integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.

Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.

The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples

Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.

Establishment and application of loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor (LAMP-LFB) for visual and rapid diagnosis of Candida albicans in clinical samples.

Candida albicans is an opportunistic pathogenic yeast that predominantly causes invasive candidiasis. Conventional methods for detecting Candida species are costly, take 3-5 days, and require skilled technicians. Rapid pathogen identification is important in managing invasive candidiasis infection. Here, a novel molecular diagnostic assay termed loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor (LAMP-LFB) was developed for C. albicans rapid detection. A set of six primers was designed based on the C. albicans species-specific internal transcribed spacer 2 (ITS2) gene. The C. albicans-LAMP results were visually reported by LFB within 2min. Various fungal strains representing Candida species, as well as several Gram-negative and Gram-positive bacterial species, were used to determine the analytical sensitivity and specificity of the assay. The optimal LAMP conditions were 64 °C for 40 min, with a sensitivity of 1fg of genomic DNA template from C. albicans pure cultures. No cross-reactions were obtained with non-albicans strains. Thus, the analytical specificity of the LAMP-LFB assay was 100%. The entire procedure could be completed within 85 min, including specimen processing (40min), isothermal reaction (40min), and result reporting (within 2min). In 330 clinical samples (including 30 whole blood, 100 middle segment urine, and 200 sputum samples), all C. albicans-positive (62/330) samples were identified by LAMP-LFB assay, and the diagnostic accuracy was 100% when compared to the traditional clinical cultural-based methods. Thus, this assay can be used as a diagnostic tool for the rapid, accurate, sensitive, low-cost and specific detection of C. albicans strains, especially in resource-limited settings.

High prevalence of Pfcrt 76T and Pfmdr1 N86 genotypes in malaria infected patients attending health facilities in East Shewa zone, Oromia Regional State, Ethiopia

BackgroundPlasmodium falciparum resistance to series of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria globally. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, Ethiopia switched from CQ to sulfadoxine–pyrimethamine (SP) and subsequently in 2004 from SP to artemether–lumefantrine (AL) for the treatment of uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons among malaria-infected patients from Adama, Olenchiti and Metehara sites of East Shewa zone, Oromia Regional State, Ethiopia.MethodsFinger-prick whole blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Extraction of parasite DNA was done by Chelex-100 method from dried blood spot (DBS). Genomic DNA template was used to amplify Pfcrt K76T and Pfmdr1 N86Y codons by nested PCR. Nested PCR products were subjected to Artherobacter protophormiae-I (APoI) restriction enzyme digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively.ResultsOf 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% carried the mutant genotypes (76T). The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti, respectively. The prevalence of Pfcrt 76T mutations in three of the study sites showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, of the 80 P. falciparum samples successfully amplified for Pfmdr1, all carried the wild-type genotypes (Pfmdr1 N86).ConclusionAlthough CQ officially has been ceased for the treatment of falciparum malaria for more than two decades in Ethiopia, greater proportions of P. falciparum clinical isolates circulating in the study areas carry the mutant 76T genotypes indicating the presence of indirect CQ pressure in the country. However, the return of Pfmdr1 N86 wild-type allele may be favoured by the use of AL for the treatment of uncomplicated falciparum malaria.

Rapid detection of 5 fungal diseases in sunflower (Helianthus annuus) using dual priming oligonucleotide system-based multiplex PCR and capillary electrophoresis.

Rapid and accurate diagnosis of fungal pathogens is essential for disease control in sunflower. In the present study, a multiplex PCR assay was developed based on the dual priming oligonucleotide (DPO) system, which was used to simultaneously detect and identify five major sunflower fungal pathogens. There was no cross-reactivity among the pathogens tested. In each reaction, 0.1 ng genomic DNA templates were sufficient to ensure specificity and accuracy. The system exhibited high adaptability over a wide range of annealing temperatures. No mismatch or nonspecific amplification was observed in the annealing temperatures tested. In combination with capillary electrophoresis, the DPO-primer-based multiplex PCR system provides a rapid, reliable and cost-efficient solution for the diagnosis of fungal pathogens in sunflower.

Development of Multiplex PCR-based Protocols for Simultaneous Caterpillar Diagnosis of Three Spodoptera and One Mamestra Species (Lepidoptera: Noctuidae).

Since many noctuid moth species are highly destructive crop pests, it is essential to establish proper management strategies, which primarily require accurate and rapid species identification. However, diagnosis of noctuid species in the field, particularly at the larval stage, is very difficult due to their morphological similarity and individual color variation. In particular, caterpillars of Spodoptera exigua (Hübner), Spodoptera litura (Fabricius), Spodoptera frugiperda (Smith), and Mamestra brassicae (L.) (Lepidoptera: Noctuidae) are hard to be identified by morphology and frequently found on the same host crops in the same season, thus requiring a reliable species diagnosis method. To efficiently diagnose these species, we identified species-specific internal transcribed spacer 1 (ITS1) sequences and developed two molecular species diagnosis protocols using ITS1 markers. The first protocol was multiplex conventional PCR in conjunction with subsequent gel electrophoresis for species identification based on amplicon size. The second protocol was based on multiplex real-time PCR using fluorescent dye-labeled primers for single-step diagnosis. Template genomic DNA (gDNA) prepared by the DNA release method was also suitable for both protocols as the template prepared by DNA extraction. The two protocols enabled rapid and robust species diagnosis using a single multiplex PCR step. Depending on laboratory instrumentation, one of the two protocols can be easily adapted for species diagnosis of the four noctuid caterpillars in the field, which is essential for establishing proper management strategies. The multiplex real-time PCR protocol, in particular, will facilitate accurate diagnosis of the four species in a single step regardless of template gDNA quality.

Detection of Haemophilus influenzae by loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay.

BackgroundHaemophilus influenzae was the most aggressive pathogen and formed a major cause of bacterial meningitis and pneumonia in young children and infants, which need medical emergency requiring immediate diagnosis and treatment. However, From isolation to identification of H. influenzae, the traditional diagnose strategy was time-consuming and expensive. Therefore, the establishment of a convenient, highly sensitive, and stable detection system is urgent and critical.ResultsIn this study, we used a combined method to detect H. influenzae. Six specific primers were designed on the basis of outer membrane protein P6 gene sequence of H. influenzae. The reaction condition such as the optimum temperature was 65℃, and the optimum reaction time was 30 min, respectively. Through the loop-mediated isothermal amplification (LAMP) in combination with nanoparticle-based lateral flow biosensor (LFB), the sensitivity of LAMP-LFB showed 100 fg was the lowest genomic DNA templates concentration in the pure cultures. Meanwhile, the specificity of H. influenzae-LAMP-LFB assay showed the exclusive positive results, which were detected in H. influenzae templates. In 55 clinical sputum samples, 22 samples were positive with LAMP-LFB method, which was in accordance with the traditional culture and Polymerase Chain Reaction (PCR) method. The accuracy in diagnosing H. influenzae with LAMP-LFB could reach 100%, compared to culture and PCR method, indicating the LAMP-LFB had more advantages in target pathogen detection.ConclusionsTaken together, LAMP-LFB could be used as an effective diagnostic approach for H. influenzae in the conditions of basic and clinical labs, which would allow clinicians to make better informed decisions regarding patient treatment without delay.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-022-02547-5.

Fabrication of Wearable PDMS Device for Rapid Detection of Nucleic Acids via Recombinase Polymerase Amplification Operated by Human Body Heat.

Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft—but tight—contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.

Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the linker sequence for the mature LASSO probe through Cre-LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (≈46% of correct probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 Escherichia coli ORFs spanning from 400- to 5000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, were able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology.

Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10−4 single cysts and single females, 4−3 single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.