[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/µg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

Specific quantitative detection of N6-methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR).

BACKGROUND: N6-methyladenosine (m6A) is a common modification in RNA, crucial for various cellular functions and associated with human diseases. Quantification of m6A at single-base resolution is of great significance for exploring its biological roles and related disease research. However, existing analysis techniques, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), face challenges like the requirement for thermal cycling or intricate primer design. Therefore, it is urgent to establish a simple, non-thermal cycling and highly sensitive assay for m6A. RESULTS: Leveraging the inhibitory effect of m6A on primer elongation and uncomplicated feature of the isothermal exponential amplification reaction (IEXPAR), we have developed an extension-based IEXPAR (E-IEXPAR). This approach requires just a single extension primer and one template, simplifying the design process in comparison to the more complex primer requirements of the LAMP methods. The reactions are conducted at constant temperatures, therby elimiating the use of thermal cycling that needed in PCR methods. By combining IEXPAR with an extension reaction, E-IEXPAR can identify m6A in RNA concentrations as low as 4 fM. We have also introduced a new analytical model to process E-IEXPAR results, which can aid to minimize the impact of unmodified adenine (A) on m6A measurements, enabling accurate m6A quantification in small mixed samples and cellular RNA specimens. SIGNIFICANCE AND NOVELTY: E-IEXPAR streamlines m6A detection by eliminating the need for intricate primer design and thermal cycling, which are common in current analytical methods. Its utilization of an extension reaction for the initial identification of m6A, coupled with a novel calculation model tailored to E-IEXPAR outcomes, ensures accurate m6A selectivity in mixed samples. As a result, E-IEXPAR offers a reliable, straightforward, and potentially economical approach for specifically assaying m6A in both biological function studies and clinical research.

Development of a PCR-based assay for specific and sensitive detection of Fusarium buharicum from infected okra plant.

Fusarium wilt, caused by the fungus Fusarium buharicum, is an emerging disease of okra in Japan. The disease was first reported in Japan in 2015, causing significant damage to okra seedlings. Due to the potential threat in okra cultivation, the development of an accurate detection method for F. buharicum is needed for the surveillance and management of the disease. In this study, we designed a primer set and developed conventional and nested PCR assays for the specific detection of F. buharicum in infected okra plants and contaminated soil, respectively. We compared the diversity of the translation elongation factor 1 alpha (EF-1α) gene of F. buharicum with 103 other fungal species/isolates to design a species-specific primer. This primer pair successfully amplified approximately 400 bp of PCR product that was only detected in the F. buharicum isolate, not in the other fungal isolates. The developed nested PCR method was highly sensitive and could detect the fungus from a 0.01 fg DNA sample. The primer successfully detected the pathogen in artificially infected plants and soil by conventional and nested PCR, respectively. This is the first report of the development of the F. buharicum-specific primer set and detection assays, which can be used for the specific and sensitive detection of F. buharicum in field samples and for taking early control measures.

A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.

Dig-up Primers: A Pipeline for Identification of Polymorphic Microsatellites Loci within Assemblies of Related Species.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers and are used in numerous fields, including conservation genetics, population genetic studies, and genetic mapping. Advances in next-generation sequencing technology and the growing amount of genomic data are driving the development of bioinformatics tools for SSR marker design. These tools work with different combinations of input data, which can be raw reads or assemblies, and with one or more input datasets. We present here a new strategy and implementation of a simple standalone pipeline that utilizes more than one assembly for the in silico design of PCR primers for microsatellite loci in more than one species. Primers are tested in silico to determine if they are polymorphic, eliminating the need to test time-consuming cross-species amplification in the laboratory. The end result is a set of markers that are in silico polymorphic in all analyzed species and have great potential for the identification of interspecies hybrids. The efficiency of the tool is demonstrated using two examples at different taxonomic levels and with different numbers of input assemblies to generate promising, high-quality SSR markers.

Integrated DNA barcoding methods to identify species in the processed fish products from Chinese market.

DNA-based methods are reliable for a precise identification of species in processed products. In this study, we assessed five typical DNA extraction methods from multiple aspects. Full-length and mini-length DNA barcoding were performed to detect the species substitution and mislabeling of 305 processed fish products from the Chinese market covering six processed fish products. The salt extraction method that exhibited the best overall performance was applied. All samples were successfully extracted; however, only 19.3 % of samples could be amplified using the full-DNA barcode primer set, and 90.2 % of samples could be amplified using the newly designed mini-DNA barcode primer sets (401 and 320 bp). Overall, the molecular identification results revealed that 36.4 % (111/305) of the samples were inconsistent with the labels, with commercial fraud observed in all six types of processed fish products. The survey findings provide technical references for effective fish authentication monitoring, offering insights into the seafood safety in markets.

Polymerase chain reaction-based methods for the rapid identification of Amanita exitialis.

Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to develop an efficient, accurate, and user-friendly PCR-based method for identifying A. exitialis that could facilitate the prevention, diagnosis, and treatment of associated food poisoning. A. exitialis-specific primers and probes were designed based on the internal transcribed spacer region variations of 27 mushroom species. Specificity was confirmed using conventional and real-time PCR for 23 non-target mushroom species, including morphologically similar and closely related species. Compared to conventional PCR, real-time PCR was more sensitive (detectable DNA concentration: 1.36 × 10-2 ng/µL vs. 1.36 × 10-3) and efficient (analysis time: 1 h vs. 40 min). Furthermore, the real-time PCR results could be immediately visualized using amplification curve analysis. The results present two robust PCR-based methods for A. exitialis identification that can facilitate food safety.

Single primer site-specific nested PCR for accurate and rapid genome-walking.

Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3' part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.

Designing Oligonucleotide-Based FISH Probe Sets with PaintSHOP.

Fluorescent in situ hybridization (FISH) enables the visualization of the position and abundance of nucleic acid molecules in fixed cell and tissue samples. Many FISH-based methods employ sets of synthetic, computationally designed DNA oligonucleotide (oligo) FISH probes, including massively multiplexed imaging spatial transcriptomics and spatial genomics technologies. Oligo probes can either be designed de novo or accessed from an existing database of pre-discovered probe sequences. This chapter describes the use of PaintSHOP, a user-friendly, web-based platform for the design of sets of oligo-based FISH probes. PaintSHOP hosts large collections of pre-discovered probes from many model organisms and also provides collections of functional sequences such as primers and readout domains and interactive tools to add these functional sequences to selected probes. Detailed examples are provided for three common experimental scenarios.

Amplidiff: an optimized amplicon sequencing approach to estimating lineage abundances in viral metagenomes.

BACKGROUND: Metagenomic profiling algorithms commonly rely on genomic differences between lineages, strains, or species to infer the relative abundances of sequences present in a sample. This observation plays an important role in the analysis of diverse microbial communities, where targeted sequencing of 16S and 18S rRNA, both well-known hypervariable genomic regions, have led to insights into microbial diversity and the discovery of novel organisms. However, the variable nature of discriminatory regions can also act as a double-edged sword, as the sought-after variability can make it difficult to design primers for their amplification through PCR. Moreover, the most variable regions are not necessarily the most informative regions for the purpose of differentiation; one should focus on regions that maximize the number of lineages that can be distinguished. RESULTS: Here we present AmpliDiff, a computational tool that simultaneously finds highly discriminatory genomic regions in viral genomes of a single species, as well as primers allowing for the amplification of these regions. We show that regions and primers found by AmpliDiff can be used to accurately estimate relative abundances of SARS-CoV-2 lineages, for example in wastewater sequencing data. We obtain errors that are comparable with using whole genome information to estimate relative abundances. Furthermore, our results show that AmpliDiff is robust against incomplete input data and that primers designed by AmpliDiff also bind to genomes sampled months after the primers were selected. CONCLUSIONS: With AmpliDiff we provide an effective, cost-efficient alternative to whole genome sequencing for estimating lineage abundances in viral metagenomes.

Direct TAMRA-dUTP labeling of M. tuberculosis genes using loop-mediated isothermal amplification (LAMP).

Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2'-deoxyuridine-5'triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs.

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Polymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer-template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer-template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different types of DNA polymerases were used. Notably, when a single-nucleotide mismatch occurred at the 3' end of the primer, we observed significant decreases in the analytical sensitivity (0-4%) with Invitrogen™ Platinum™ Taq DNA Polymerase High Fidelity, while the analytical sensitivity remained unchanged with Takara Ex Taq Hot Start Version DNA Polymerase. Leveraging these findings, we designed a highly specific PCR to amplify Babesia while effectively avoiding the genetically close Theileria. Through elucidating the critical interplay between types of DNA polymerases and primer-template mismatches, this research provides valuable insights for improving PCR accuracy and performance. These findings have important implications for researchers aiming to achieve robust qPCR results in various molecular biology applications.

Gated nanoprobe utilizing metal-organic frameworks for identifying and distinguishing between the wild strains and the vaccine strains of brucella.

In the assay for Brucella, the identification and differentiation of wild strains and vaccine strains present a significant challenge. Currently, there aren't any commercially available product to address this issue. In this study, we have developed a novel gated nanoprobe by utilizing Metal-Organic Frameworks (MOFs) as a scaffold and hairpin DNA as a "gating switch". Specifically, Probe 1 with hairpin structure (P1h) targets a gene that is present in both wild strains Y3 (B. melitensis biovar 3) and vaccine strains A19 (Brucella abortus strains A19). We successfully applied this probe to screen positive samples of Brucella without any cross-reactivity with other substances. Additionally, we identified another specific gene exclusively found in wild strains, which serves as Probe 2 with hairpin structure (P2h) to confirm the strain type. Simultaneous detachment of both P1h and P2h from the MOFs leads to the release of Rhodamine 6G (Rho 6G) and Fluorescein (Flu), specifically indicating the presence of wild strains. If only P1h detaches and the Flu signal is detected, it suggests the presence of vaccine strains. Importantly, this method offers high accuracy, with a detection rate of 90% and a recovery rate of 94.71% to 107.65%, while avoiding cross-reactions with MO and TB. This one-step experiment provides reliable identification and differentiation of Y3 and A19, addressing concerns related to long periodicity, interference from individual variations, and the complex design of primers in existing laboratory methods. Furthermore, our approach successfully detects target 1 (T1) and target 2 (T2) at concentrations ranging from 10-6 M to 10-9 M, with a detection limit of 6.7 × 10-10 M and 6.4 × 10-10 M, respectively. Importantly, our strategy is cost-effective (around $1) and offers higher detection efficiency compared to traditional laboratory methods.

CRISPR/Cas12a integrated electrochemiluminescence biosensor for pufferfish authenticity detection based on NiCo2O4 NCs@Au as a coreaction accelerator.

Meat adulteration has brought economic losses, health risks, and religious concerns, making it a pressing global issue. Herein, combining the high amplification efficiency of polymerase chain reaction (PCR) and the accurate recognition of CRISPR/Cas12, a sensitive and reliable electrochemiluminescence (ECL) biosensor was developed for the detection of pufferfish authenticity using NiCo2O4 NCs@Au-ABEI as nanoemitters. In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a is activated upon specific recognition by crRNA, and then it cleaves dopamine-modified single stranded DNA (ssDNA-DA), triggering the ECL signal from the "off" to "on" state. However, without target DNA, the trans-cleavage activity of CRISPR/Cas12a is silenced. By rationally designing corresponding primers and crRNA, the biosensor was applied to specific identification of four species of pufferfish. Furthermore, as low as 0.1 % (w/w) adulterate pufferfish in mixture samples could be detected. Overall, this work provides a simple, low-cost and sensitive approach to trace pufferfish adulteration.

Protocol to retrieve unknown flanking DNA sequences using semi-site-specific PCR-based genome walking.

Here, we describe a protocol based on semi-site-specific primer PCR (3SP-PCR) to access unknown flanking DNA sequences. We specify the guidelines for designing primers for 3SP-PCR. We also describe experimental procedures for the 3SP-PCR, along with PCR product purification and subsequent sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Wei et al.1.

Short-stranded DNA segment-modulated LAMP/H+ as signal transducer to guide CHA-cooperated amplifiable electrochemical biosensing.

BACKGROUND: Modulating loop-mediated isothermal amplification (mLAMP) by short-stranded DNA segment trigger (T) to generate byproducts H+ ions (mLAMP/H+) as signal transducer is intriguing for developing catalytic hairpin assembly (CHA)-cooperated amplifiable electrochemical biosensors. This would be a big challenge for traditional LAMP that is basically suitable for amplifying long-stranded oligonucleotides up to 200-300 nt. To address this inherent limitation of traditional LAMP, many researchers have put in efforts to explore improvements in this that would allow LAMP to be used for a wider range of target species amplification. RESULTS: Here in this work, we are inspired to explore two-step loop-mediated amplification, firstly forming T-activated double-loop dumbbell structure (DLDS) intermediate by a recognition hairpin and a hairpin precursor, and next DLDS-guided mLAMP process with the aid of two primers to yield mLAMP/H+ during successive DNA incorporation via nucleophilic attacking interaction. To manipulate the mLAMP/H+-directed transduction of input T, a pH-responsive triplex strand is designed with the ability of self-folding in Hoogsteen structure at slightly acidic conditions, resulting in the dehybridization of a fuel strand (FS) to participate in CHA between two hairpins on the modified electrode surface, in which FS is repetitively displaced and recycled to fuel the progressive CHA events. In the as-assembled dsDNA complexes, numerous electroactive ferrocene labels are immobilized in the electrode sensing interface, thereby generating significantly amplified electrochemical current signal that can sense the presented and varied T. SIGNIFICANCE: It is clear that we have creatively constructed a unique electrochemical biosensor for disease detection. Benefited from the rational combination of mLAMP and CHA, our electrochemical strategy is highly sensitive, specific and simplified, and would provide a new paradigm to construct various mLAMP/H+-based biosensors for other short-stranded DNA or microRNAs markers.

Integration of IFAST-based nucleic acid extraction and LAMP for on-chip rapid detection of Agroathelia rolfsii in soil.

Agroathelia rolfsii (A. rolfsii) is a fungal infection and poses a significant threat to over 500 plant species worldwide. It can reduce crop yields drastically resulting in substantial economic losses. While conventional detection methods like PCR offer high sensitivity and specificity, they require specialized and expensive equipment, limiting their applicability in resource-limited settings and in the field. Herein, we present an integrated workflow with nucleic acid extraction and isothermal amplification in a lab-on-a-chip cartridge based on immiscible filtration assisted by surface tension (IFAST) to detect A. rolfsii fungi in soil for point-of-need application. Our approach enabled both DNA extraction of A. rolfsii from soil and subsequent colorimetric loop-mediated isothermal amplification (LAMP) to be completed on a single chip, termed IFAST-LAMP. LAMP primers targeting ITS region of A. rolfsii were newly designed and tested. Two DNA extraction methods based on silica paramagnetic particles (PMPs) and three LAMP assays were compared. The best-performing assay was selected for on-chip extraction and detection of A. rolfsii from soil samples inoculated with concentrations of 3.75, 0.375 and 0.0375 mg fresh weight per 100-g soil (%FW). The full on-chip workflow was achieved within a 1-h turnaround time. The platform was capable of detecting as low as 3.75 %FW at 2 days after inoculation and down to 0.0375 %FW at 3 days after inoculation. The IFAST-LAMP could be suitable for field-applicability for A. rolfsii detection in low-resource settings.

Development and Testing of Species-Specific Primers for Detecting the Presence of the Northern Pacific Sea Star (Asterias amurensis) from Environmental DNA.

The starfish Asterias amurensis, a well-known predator of molluscan species in intertidal ecosystems, has caused substantial ecological and economic losses in North China such as offshore Qingdao. Effective monitoring and prevention measures are urged to minimize its negative impacts. Compared with traditional biomonitoring methods, environmental DNA technology has emerged as a powerful and cost-efficient tool for inferring species' presence and abundance. In this study, we developed a pair of species-specific primers (i.e., Ast-F and Ast-R) for the A. amurensis mitochondrial COI gene and tested its utility in amplifying and quantifying the DNA fragments from environmental samples under both laboratory and field conditions. The results of controlled water tank experiments demonstrated that the amount of eDNA released by A. amurensis was positively related to its biomass; after the removal of the starfish, the eDNA degraded significantly in 24 h and remained detectable for 8 days. The number of eDNA copies enriched tended to increase with smaller pore size of filter membrane and larger volume of filtered water. For field tests, we confirmed the validation of our approach in six locations in Qingdao by filtering 1000 ml water per sample with a 0.45-µm pore size filtration. All the amplification products generated a single and bright band via gel electrophoresis, and the quantitative PCR results unveiled significant differences in eDNA copies. This study provided an eDNA-based approach for investigating the distribution and biomass of A. amurensis, which may help to formulate early warning and management strategies in coastal Qingdao and other regions.

Comparison of pre-labelled primers and nucleotides as DNA labelling method for lateral flow detection of Legionella pneumophila amplicons.

Labelling of nucleic acid amplicons during polymerase chain reaction (PCR) or isothermal techniques is possible by using both labelled primers and labelled nucleotides. While the former is the widely used method, the latter can offer significant advantages in terms of signal enhancement and improving the detection limit of an assay. Advantages and disadvantages of both methods depend on different factors, including amplification method, detection method and amplicon length. In this study, both methods for labelling PCR products for lateral flow assay (LFA) analysis (LFA-PCR) were analysed and compared. It was shown that labelling by means of nucleotides results in an increase in label incorporation rates. Nonetheless, this advantage is negated by the need for post-processing and competitive interactions. In the end, it was possible to achieve a detection limit of 3 cell equivalents for the detection of the Legionella-DNA used here via primer labelling. Labelling via nucleotides required genomic DNA of at least 3000 cell equivalents as starting material as well as an increased personnel and experimental effort.