DNA Amplification Research Articles

Harnessing the potential of transverse thin cell layer culture for high-frequency micropropagation of Thai ginseng (Kaempferia parviflora Wall. Ex Baker)

In vitro plant propagation techniques play an important role in modern agriculture and horticulture, offering efficient methods for the accelerated multiplication of high-value plant species. Thin cell layer (TCL) culture technique has emerged as a promising approach for large-scale in vitro plantlets production. This study explores the potential of TCL culture as a cutting-edge strategy to enhance the efficiency of multiple shoots formation in Kaempferia parviflora. An attempt was also made to look into the synergistic effects of cytokinin and auxin [6-benzyladenine (BA) - indole-3-acetic acid (IAA)] during micropropagation. Shoot tip explants (from rhizome sprouts) were cultured onto Murashige and Skoog (MS) medium enriched with BA (2.0–8.0 mg/L) and IAA (0.2–0.8 mg/L) in combination where the highest shoot number (7.3 shoots/explant) and length (5.9 cm) was obtained in 8.0 mg/L BA and 0.8 mg/L IAA. Subsequently, the transverse thin cell layer (tTCL) culture system was successfully utilized whereby the shoot tip explants were individually sliced from tips at the meristem and cultured onto MS medium supplemented with BA (2.0–8.0 mg/L) and IAA (0.2–0.8 mg/L) in combination. The regenerated tTCLs exhibited the highest shoot multiplication (9.3 shoots/tTCL; 28 shoots/shoot tip) and length (5.3 cm) in 4.0 mg/L BA + 0.8 mg/L IAA, thus significantly increased number of plantlets were obtained from tTCL culture (953.0 plantlets) compared to shoot tip culture (527.7 plantlets) after six sub-culture cycles. The tTCL regenerants were successfully acclimatized (with 90% survival) and displayed active growth. Genetic fidelity assessment via inter-simple sequence repeats, conserved DNA-derived polymorphism, directed amplification of minisatellite-region DNA, and start codon targeted polymorphism marker systems suggested that a very low level (5.8%; 148 out of 2572 scorable bands) of polymorphism was detected whereas the ploidy level remained unchanged, confirmed by flow cytometry analysis. The implementation of tTCL culture, coupled with a controlled sub-culture regimen, holds significant potential for the efficient commercial-scale production of K. parviflora.

New metagenomic procedure for the investigation of the eukaryotes present in the digestive gland of Mytillus galloprovincialis

Mussels’ aquaculture is one of the most important cultures in Europe, with a high level of self-sufficiency. In Spain, the main cultivated species is M. galloprovincialis, whose production takes place primarily in Galicia. In that region, a Protected Designation of Origin certification exists to certify the high quality of the product and its sustainability. This species is also present in other regions making the control of its traceability complicated. In order to distinguish Galician mussels from the others, the eukaryotes present in its digestive gland could be a useful tool. However, to use them as biomarkers, having an adapted protocol to recover the diversity present in the digestive gland is essential. In this work, we have selected a new protocol to optimize the study of the diversity of eukaryotes associated with the digestive gland of M. galloprovincialis. This protocol is based on a combination of a pre-treatment and a commercial DNA extraction kit, the amplification of DNA with two newly designed primer sets to avoid the DNA from the mussel, and the use of next-generation sequencing. The results showed a significant reduction of mussel DNA amplification and enough DNA quality for the subsequent sequencing, increasing the number of sequences recovered from other eukaryotes. This new protocol offers then a good chance to determine the diversity of eukaryotes present in the digestive gland of M. galloprovincialis, and it can be used in future studies to examinate the seasonal variation of the present species and to evaluate its potential as geographical traceability tool.

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

Mammal dung-dung beetle trophic networks: an improved method based on gut-content DNA.

Dung beetles provide many important ecosystem services, including dung decomposition, pathogen control, soil aeration, and secondary seed dispersal. Yet, the biology of most dung beetles remains unknown. Natural diets are poorly studied, partly because previous research has focused on choice or attraction experiments using few, easily accessible dung types from zoo animals, farm animals, or humans. This way, many links within natural food webs have certainly been missed. In this work, we aimed to establish a protocol to analyze the natural diets of dung beetles using DNA gut barcoding. First, the feasibility of gut-content DNA extraction and amplification of 12s rDNA from six different mammal dung types was tested in the laboratory. We then applied the method to beetles caught in pitfall traps in Ecuador and Germany by using 12s rDNA primers. For a subset of the dung beetles caught in the Ecuador sampling, we also used 16s rDNA primers to see if these would improve the number of species we could identify. We predicted the likelihood of amplifying DNA using gut fullness, DNA concentration, PCR primer, collection method, and beetle species as predictor variables in a dominance analysis. Based on the gut barcodes, we generated a dung beetle-mammal network for both field sites (Ecuador and Germany) and analyzed the levels of network specificity. We successfully amplified mammal DNA from dung beetle gut contents for 128 specimens, which included such prominent species as Panthera onca (jaguar) and Puma concolor (puma). The overall success rate of DNA amplification was 53%. The best predictors for amplification success were gut fullness and DNA concentration, suggesting the success rate can be increased by focusing on beetles with a full gut. The mammal dung-dung beetle networks differed from purely random network models and showed a moderate degree of network specialization (H2': Ecuador = 0.49; Germany = 0.41). We here present a reliable method of extracting and amplifying gut-content DNA from dung beetles. Identifying mammal dung via DNA reference libraries, we created mammal dung-dung beetle trophic networks. This has benefits over previous methods because we inventoried the natural mammal dung resources of dung beetles instead of using artificial mammal baits. Our results revealed higher levels of specialization than expected and more rodent DNA than expected in Germany, suggesting that the presented method provides more detailed insights into mammal dung-dung beetle networks. In addition, the method could have applications for mammal monitoring in many ecosystems.

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status

BackgroundAnalysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. ResultsMBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. Significance and noveltyReal-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

Evaluation of Pre-Analytical Variables for Human Papillomavirus Primary Screening from Self-Collected Vaginal Swabs

HPV primary screening is an effective approach to assessing cervical cancer risk. Self-collected vaginal swabs have shown promise to expand testing access, but there are limited data defining analytical performance criteria necessary for adoption of self-collected specimens, especially for those occurring outside the clinic where the swab remains dry during transport. Here, we evaluated the performance of self-collected vaginal swabs for HPV detection using the Roche Cobas 6800. There was insignificant variability between swabs self-collected by the same individual (n=15 participants collecting 5 swabs/participant), measured by amplification of HPV and human β-globin control DNA. Comparison of self-collected vaginal swab and provider-collected cervical samples (n=144 paired collections) proved highly concordant for HPV detection (total agreement=90.3%;PPA=84.2%). There was no relationship between the number of dry storage days and amplification of HPV (n=68, range 4-41days). Exposure of self-collected dry swabs to extreme summer and winter temperatures did not affect testing outcomes. We assessed a second internal control (RNAseP) in a subset and demonstrate that lack of amplification for β-globin from self-collected specimens was consistent with poor, but not absent, cellularity. These data suggest that self-collected vaginal samples enable accurate clinical HPV testing, and that extended ambient dry storage or exposure to extreme temperatures do not influence HPV detection. Further, lack of β-globin amplification in HPV negative samples accurately identified participants that required recollection.

DNA Profiling, Bioinformatics and Databases in Forensics: Human Identification Purposes

Individual identification is an essential element in establishing truth the forensic investigation process, be it in criminal, medico-legal, or mass disasters case. When ante-mortem data are not available, the only thing that can be used is identification trough DNA. Alec Jeffrey, a geneticist (1984), found that certain regions of DNA contain repetitive sequences and the number of repetitions in individuals differs from one another. This identification method known as DNA profiling. DNA profiling is described as an important and significant discovery in forensics and has been considered as the standard in modern human identification. Until now, the recommended DNA profiling method must be based on the PCR to analyze degraded DNA and short-sized DNA (Short Tandem Repeats) through PCR amplification. In profiling, the result DNA amplification are analyzed through genetic markers (DNA Markers) and then matched into the database or compared with the collected reference samples. In the human genome, the genetic markers most frequently used in forensics are autosomal STR, Y-STR, mtDNA, X-STR, SNPs, and Amelogenin. Accomplishment of proving the suitability of DNA profiles, an application of bioinformatics in forensics is carried out. Bioinformatics is a combination of molecular biology and computer informatics that aims to manage and analyze data and store biological (genetic) information. DNA database is an application of bioinformatics in the form of special software which has become an important tool for biologists and forensics. This genetic database will predict the similarities between one DNA profile and another.

Isothermal Reciprocal Catalytic DNA Circuit for Sensitive Analysis of Kanamycin.

Inappropriate use of veterinary drugs can result in the presence of antibiotic residues in animal-derived foods, which is a threat to human health. A simple yet efficient antibiotic-sensing method is highly desirable. Programmable DNA amplification circuits have supplemented robust toolkits for food contaminants monitoring. However, they currently face limitations in terms of their intricate design and low signal gain. Herein, we have engineered a robust reciprocal catalytic DNA (RCD) circuit for highly efficient bioanalysis. The trigger initiates the cascade hybridization reaction (CHR) to yield plenty of repeated initiators for activating the rolling circle amplification (RCA) circuit. Then the RCA-generated numerous reconstituted triggers can reversely stimulate the CHR circuit. This results in a self-sufficient supply of numerous initiators and triggers for the successive cross-invasion of CHR and RCA amplifiers, thus leading to exponential signal amplification for the highly efficient detection of analytes. With its flexible programmability and modular features, the RCD amplifier can serve as a universal toolbox for the high-performance and accurate sensing of kanamycin in buffer and food samples including milk, honey, and fish, highlighting its enormous promise for low-abundance contaminant analysis in foodstuffs.

Seroprevalence and molecular investigation of toxocariasis in working children and control group in Tehran, Iran

BackgroundToxocariasis, a zoonosis helminthic disease is caused in humans by Toxocara canis and Toxocara cati with worldwide distribution. In this study the seroprevalence and molecular toxocariasis were investigated in the working children and the control group in Tehran, Iran. MethodsIn total, 468 children between the ages of 7–14 years old, including 280 blood samples from working children and 188 samples from control group children were collected from Tehran. ELISA test was performed to detect IgG antibody against Toxocara canis. Molecular tests using ITS2 primer were performed to determine the status of toxocariasis infection in children. ResultsThe seroprevalence of toxocariasis was 24 (8.6%) and 10 (5.3%) in the working children and control group respectively. Seroprevalence of toxocariasis in girls 18(13.5%) was higher than boys 6(4.1%) in working children and differences was statistically significant. A significant relationship was found between contact with soil or contact with cats and dogs and the seroprevalence of toxocariasis in working children. No amplification of the target DNA was detected in plasma or whole blood samples in working children and the control group with Toxocara canisand cati primers. ConclusionThe seroprevalence of IgG antibody against Toxocara canis was higher in working children than in the control group in Tehran. The result of the present study indicated a significant difference between working children and the control group in terms of Toxocara canis antibody titer. More contact with soil in these children and contact with dogs and cats may be factors in acquiring toxocariasis in the children.

Evaluation of molecular inhibitors of loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification (LAMP) is a cost-effective and easy-to-perform assay that enables the direct detection of DNA. Its use in point-of-care diagnostic tests is growing, while it has the potential to be used in presumptive on-the-field forensic tests. Samples are often collected from complex matrices that contain high levels of contaminants. Herein, we evaluate the effect of seven common DNA amplification inhibitors on LAMP – bile salts, calcium chloride, hematin, humic acid, immunoglobulin G, tannic acid and urea. We study the effect of each inhibitor individually in real-time detection systems coupled with end-point measurements to delineate their inhibitory effects from the matrix in which they may be found. Our studies show LAMP inhibitors generally delay the onset of amplicon formation and quench fluorescence at similar or higher concentrations compared to PCR, but that end-point measurements of LAMP amplicons are unaffected. This is important as LAMP amplicons can be detected in non-fluorometric ways thus contributing to the assertions that LAMP is more robust to inhibitors than PCR.

Bacteriophage-assisted lysis and eluted genomic DNA-based detection of pathogenic bacterial contamination in food

Foodborne diseases represent a significant global health concern, with a substantial number of illnesses and fatalities reported annually owing to the consumption of contaminated food. Current methods for identifying foodborne pathogens cannot effectively distinguish live from dead bacteria or efficiently detect multiple targets. Therefore, we developed a selective detection method for three pathogenic bacteria in food samples that leverages bacteriophage-assisted live-cell lysis and exposed genomic DNA amplification. The antimicrobial activity and host specificity of these three bacteriophages were employed to confirm the target bacteria selectivity. Bacterial DNA purification using bacteriophage-assisted lysis demonstrated the superior activity of the selected phages in extracting DNA from Escherichia coli O157:H7 and Salmonella Typhimurium. The results from reactions involving the three strains, including Bacillus cereus, highlighted that a 1 h incubation period yielded the most efficient DNA extraction and purification. The polymerase chain reaction (PCR) products obtained were comparable in terms of concentration and purity with those generated using bacterial DNA prepared using a commercial kit. In the practical food sample application experiment, bacterial strains were successfully recovered and concentrated using a polyethersulfone filter, resulting in a concentration factor >3 for all samples. Furthermore, the concentration of purified bacterial DNA from contaminated kimchi cabbage exceeded 10 ng/μL. Notably, we could detect E. coli O157:H7, S. Typhimurium, and B. cereus spiked at 103 colony-forming units/mL using multiplex PCR. This innovative bacteriophage-based approach holds substantial promise for the selective and practical detection of live foodborne pathogens, thereby significantly advancing food safety standards.

Short Communication: Analysis of simple sequence repeats variations for the assessment of genotypic diversity in Centella asiatica (L.) Urb.

Abstract. Maruzy A, Susandarini R. 2024. Short Communication: Analysis of simple sequence repeats variations for the assessment of genotypic diversity in Centella asiatica (L.) Urb. Biodiversitas 25: 734-740. Centella asiatica (L.) Urb. is a perennial plant widely known as herbal ingredient in traditional and modern medicine, as well as in the cosmetics industry. This herbaceous species grows in various types of habitat, and is known for its morphological variations among populations, indicating the existence of genotypic variation. In this study, the assessment of genotypic diversity using simple sequence repeats (SSR) was carried out on C. asiatica accessions from 32 populations collected from eight mountains in Central Java. Representative individuals from four populations grow at different altitudes were sampled in each mountain. Amplification of genomic DNA using the SSR primer mCaCIR002 produced a single amplicon of 180 bp. The results of SSR analysis showed that there is high genotypic diversity in C. asiatica which is characterized by variations in the number of microsatellite repeats of (CT)10 to (CT)19. Variations in the number of repeats are mainly caused by insertions and deletions in DNA sequences at the positions 102-145. Haplotype analysis also showed high genetic diversity within and between populations of C. asiatica, indicated by the haplotype diversity of 1.00 in all populations. The haplotype network analysis indicated that there was no gene flow between the four populations at each mountain where C. asiatica populations grow. The results of this study provide evidence for the role of SSR as a suitable molecular marker for studying genotypic diversity at the intraspecific level, and confirming the morphological variability in this species.

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing

Control of CRISPR/Cas12a trans-cleavage is crucial for biosensor development. Here, we show that small circular DNA nanostructures which partially match guide RNA sequences only minimally activate Cas12a ribonucleoproteins. However, linearizing these structures restores activation. Building on this finding, an Autocatalytic Cas12a Circular DNA Amplification Reaction (AutoCAR) system is established which allows a single nucleic acid target to activate multiple ribonucleoproteins, and greatly increases the achievable reporter cleavage rates per target. A rate-equation-based model explains the observed near-exponential rate trends. Autocatalysis is also sustained with DNA nanostructures modified with fluorophore-quencher pairs achieving 1 aM level (<1 copy/μL) DNA detection (106 times improvement), without additional amplification, within 15 min, at room temperature. The detection range is tuneable, spanning 3 to 11 orders of magnitude. We demonstrate 1 aM level detection of SNP mutations in circulating tumor DNA from blood plasma, genomic DNA (H. Pylori) and RNA (SARS-CoV-2) without reverse transcription as well as colorimetric lateral flow tests of cancer mutations with ~100 aM sensitivity.

A Fusion of Taq DNA Polymerase with the CL7 Protein from Escherichia coli Remarkably Improves DNA Amplification.

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.

DNA and ATP synergistically triggered Argonaute-mediated sensor for the ultrasensitive detection of viable Salmonella without DNA extraction and amplification

Foodborne pathogens present a significant threat to human health. In this study, a phage-assisted DNA and ATP synergistically triggered Argonaute-mediated fluorescent (PDAAF) biosensor was developed for the ultrasensitive detection of viable Salmonella with DNA extraction-free and amplification-free. The phage can lyse viable Salmonella to release DNA and ATP, which can reflect the amount of bacteria present. First, the two-step cleavage ability of Pyrococcus furiosus Argonaute (PfAgo) was used to identify the target DNA and cleave fluorescent probes. Second, the bivalent aptamer recognized the ATP and released guide DNA that combined with PfAgo to cleave fluorescent probes. The DNA and ATP synergistically triggered double cleavage in a single tube, greatly enhancing the detection sensitivity for Salmonella (limit of detection was 20 CFU/mL). The spiked recoveries (87.72%-113.97%) and real sample analysis demonstrated that the PDAAF biosensor possesses good accuracy and applicability, effectively enabling the DNA extraction- and amplification-free detection of foodborne pathogens.

Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta.

Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L-1. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA-LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10-1 ng μL-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry.

First report of the maize cyst nematode Heterodera zeae in Sichuan Province of Southwest China.

Maize is the largest crop planted in China. Nine species of cyst nematodes have been reported to affect maize production. Heterodera zeae, H. avenae and Punctodera chalcoensis can cause significant maize yield losses annually (Luc et al. 2005). In 1971, the maize cyst nematode H. zeae was first detected in Rajasthan, India (Koshy et al. 1971). Subsequently, it has been reported in many other countries such as the United States, Greece, Pakistan, and Egypt. In China, H. zeae was first identified in the maize fields of Laibin City, Guangxi Zhuang Autonomous Region (Wu et al., 2017). Cui et al. (2020) identified H. zeae in a maize field of Yuzhou City, Henan Province of Central China in 2018. From 2018 to 2022, a survey of cyst-forming nematodes was conducted in Southwest China. Fifteen soil samples of about 500 g each were collected from Luding County, Ganzi Prefecture of Sichuan Province. No major aboveground symptoms were shown on maize, but a few females were observed on the roots of maize in one field. The cysts and second-stage juveniles (J2s) were collected from each soil sample using Cobb's screening gravity method. A total of 8.50±2.0 cysts per 100 ml of soil on the average were observed in the field. A thin subcrystalline layer was discernible only in young cysts. Morphological and molecular studies of cysts and J2s indicated that the nematodes were identified to be H. zeae in a maize-field. Morphologically, the cysts were in a lemon shape, light brown or pearly white in color. The vulval cone was prominent. Fenestra ambifenestrate, and semifenestra were separated by a fairly wide vulval bridge, fenestral length and width were variable, and the cyst wall was shown in a zigzag pattern. The J2s' body was in a vermiform, tapering at both ends, with a hyaline tail. Stylet was strongly developed with round or slightly anteriorly directed knobs. Morphological measurements of the cysts (n = 9) determined that the mean body length was 417.2 μm (403.6 to 439.4 μm), body width was 429.7 μm (397.6 to 456.9μm); length-width ratio was 1.4 (0.75 to 3); fenestra length was 525.3 μm (498.5 to 570.7 μm); and the mean semifenestra width was 458.6 μm (403.6 to 546.3 μm). Morphometric measurements of second-stage juveniles (n = 20) showed a body length of 419.7μm (355.8 to 492.5 μm); a stylet length of 20.8 μm (19.51 to 23.3μm); a tail length of 41.5 μm (20 to 49.4 μm); and a hyaline tail length of 20.7 μm (16.6 to 24 μm). The main morphological characteristics and measured values were basically consistent with those described by Cui et al. (2022), and all of which were similar to those of H. zeae. Amplification of DNA from random single cysts (n = 5) was conducted using the protocol described by Cui et al. (2022). The rDNA-internal transcribed spacer (ITS) was amplified and sequenced using a pair of universal primers TW81 (5'-GTTTCCGTAGGTGAA CCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3'). The ITS sequences were deposited at GenBank with the accession number OR811029.1. Alignments of sequences showed an identity of 98% with H. zeae sequences from China (OP692769.2, MW785772.1) and the USA (GU145616.1), which were confirmed using a pair of species-specific primers HzF1 (5'-GGGGAGGTGAATGTGGG-3') and HzR1 (5'-CCTTTGGCAATCGGTGA-3') of H. zeae with a targeted PCR fragment of 393 bp (Cui et al. 2022). Pathogenicity was conducted and confirmed by infection and reproduction on maize. Seeds (cv. Zhengda 619) were sown in three pots that contained 150 ml of a sterile soil mixture (loamy soil: sand=1:1), and 5 cysts (103 eggs/cyst on the average) were inoculated in each pot at 25/30°C, under a 12-h dark/12-h light condition (Cui et al. 2023). Fifteen days after sowing, third- and fourth-stage juveniles were observed in the rootstained with acid fuchsin, and a total of 32 cysts per maize plant on the average were collected at 40 days after sowing. The new cysts' morphological and molecular characteristics were identical to the cysts from the original soil samples. To the best of our knowledge, this is the first report of H. zeae as a pathogen on maize in Sichuan Province, Southwest China. Our findings will be useful for management and further research of maize cyst nematodes.

Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR.

Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.

A novel loop-mediated isothermal amplification (LAMP) assay to diagnose feline panleukopenia.

Protoparvovirus carnivoran1, known commonly as feline panleukopenia virus (FPV) is a highly contagious and environmentally stable parvovirus of domestic as well as wild felids. A rapid and robust diagnostic tool will aid in implementing prompt treatment and control measures. A loop-mediated isothermal amplification (LAMP) as a point-of-care diagnostic tool for diagnosing feline panleukopenia was standardised using faecal samples of cats. The assay will reduce the cost and time required to diagnose feline panleukopenia. A set of two outer primers (F3 and B3) and two inner primers (FIP and BIP) were designed to target the viral polypeptide (VP2) gene of FPV. Optimisation of the LAMP reaction was done at 60 °C for one hour after an initial denaturation at 95 °C for five minutes. Visualisation of the result based on the addition of SYBR Green 1 dye offered an easy and reliable diagnosis. The detection limit of the standardised LAMP assay was as low as 1.25 ng/μl of the target DNA. Species specificity of the LAMP primers revealed no amplification of the non-target DNA of any other species except that of the canine parvovirus DNA template. DNA extracted from 100 PCR-positive and 20 PCR-negative faecal samples were subjected to the standardised assay and compared with PCR. Analysis of the results revealed that the LAMP assay was 100% sensitive and 90% specific compared to PCR. The LAMP assay could be a reliable tool for the point-of-care diagnosis of feline panleukopenia in limited resource settings.