Introduction

Veillonella spp. are Gram-negative, obligate anaerobes that are present in the oral cavity and form part of the normal flora of mammalian organisms (Yuan et al. 2021). To date, 15 species of Veillonella (V. atypica, V. caviae, V. criceti, V. denticariosi, V. dispar, V. infantium, V. magna, V. montpellierensis, V. nakazawae, V. parvula, V. ratti, V. rodentium, V. rogosae, V. seminalis, V. tobetsuensis) have been reported, and detailed information on their isolation and pathogenicity is listed in Table 1. The majority of these species have been detected in humans, with only three species, namely V. caviae, V. criceti, and V. magna, being reported only in animals to date (Fu et al. 2022; Jiang et al. 2016; Kraatz and Taras 2008). However, it is worth noting that V. ratti was also previously thought to be present only in animals, but was later detected in the human intestine and reproductive tract (Marchandin et al. 2005; Pham et al. 2017).

Table 1 Information on the isolation and pathogenicity of Veillonella
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Veillonella spp. share similar properties, including utilizing lactic acid, in preference to carbohydrates, as an energy source in the host organism. On the one hand, the lactic acid produces short-chain fatty acids, such as acetic acid and propionic acid, to maintain the health of the organism (Mashima et al. 2021), and on the other hand, it produces toxic LPS and H2S, which pose a risk of disease in immunocompromised individuals (Zhan et al. 2022). The specific metabolites of Veillonella have led to controversy among researchers regarding the outcome of their elevated abundance. Some studies have suggested that the elevated abundance of lactic acid-using Veillonella may decrease lactate accumulation during exercise and thus elevate the body’s endurance (Scheiman et al. 2019). However, many studies have suggested that elevated abundance of Veillonella can cause a range of diseases. For example, V. parvula has been detected in blood, bone, the oral cavity, urinary tract, heart valves, cerebrospinal fluid, respiratory tract, and gastrointestinal tract, causing multiple diseases including bacteremia, intervertebral discitis, dental caries, urinary tract infection, endocarditis, meningitis, and inflammatory bowel disease (IBD) (Chang et al. 2023; Rojas-Tapias et al. 2022; Li et al. 2017; Bhatti and Frank 2000; Ke et al. 2022; Berenger et al. 2015; Chen et al. 2021; Washio et al. 2014). Under normal conditions, Veillonella is not present in the blood, bones, heart valves, or cerebrospinal fluid, and if detected at these sites, it is considered a potential pathogen. However, in the oral cavity, respiratory tract, and gastrointestinal tract, where Veillonella is already present under natural conditions, its level of abundance is critical to the disease state. The quantification of Veillonella abundance in patient samples by molecular techniques is therefore of great significance for disease diagnosis.

Previous studies involving the molecular detection of Veillonella were mainly focused at the species level. For example, Rôças and Siqueira Jr (2006) reported the detection of V. parvula by nested PCR, and Mashima and Nakazawa reported the detection of V. tobetsuensis and V. infantium (Mashima and Nakazawa 2013; Mashima et al. 2018a) by amplification of the DnaK gene. Although the specificity of species-level detection is high, the detection spectrum is narrow in clinical practice and difficult to apply to the detection of samples containing unknown pathogens. Rinttilä et al. proposed a qPCR assay for the detection of Veillonella at the genus level, but the specificity was poor, partly because the assay system incorporated an insufficient number of strains (Rinttilä et al. 2004). A new detection method for Veillonella is therefore required. In this study, we designed a pair of primers and a probe that were highly conserved within, and highly specific to, genera to establish a rapid and objective quantitative assay involving multiplexed sequence alignment. We then analyzed the applicability of this assay using clinical samples.

Materials and methods

Ethical approval

This study was approved by the medical ethics committee of the capital institute of pediatrics, Bei**g, China.

Bacterial strains

Four species of Veillonella and seven common clinical pathogens were used to evaluate the test system, including V. dispar, V. nakazawae, V. parvula, V. rodentium, Enterococcus faecalis, Klebsiella pneumoniae, Morganella morganii, Escherichia coli, Proteus mirabilis, Salmonella enteritidis, and Shigella dysenteriae. V. parvula (ATCC 10790), V. Nakazawa (CCUG 74597T), V. dispar (ATCC 17745), and V. rodentium (ATCC 17743) are the standard strains, and the rest of the strains have been identified and stored in the Bacteriology Research Laboratory of the Capital Institute of Pediatrics (Feng et al. 2023).

Strain culture and DNA extraction

V. dispar, V. nakazawae, V. parvula, and V. rodentium were incubated for 48 h under anaerobic conditions at 37 °C in brain heart infusion (BHI) supplemented with 5% defibrinated sheep blood and 2% sodium lactate. E. faecalis, K. pneumoniae, M. morganii, E. coli, P. mirabilis, S. enteritidis, and S. dysenteriae were incubated for 24 h under aerobic conditions at 37 °C in BHI.

The bacterial solution was collected from the cultured bacteria, and the optical density of it measured at a wavelength of 600 nm (OD600) was monitored by spectrophotometer, and the OD600 was adjusted to 1.0 with PBS buffer. The total bacterial DNA was extracted from the collected bacterial solution and stored at −80 °C according to the instruction of TIANamp Bacteria DNA Kit (TIANGEN, China).

Multiple sequence matching

The primer and probe were designed at the genus level according to the 16S rRNA gene sequences. The 16S rRNA gene data of 15 species of Veillonella were downloaded from EZbiocloud database, and the gene sequences of 6 other genera of Veillonellaceae and 9 common genera of the intestine were also searched, and the corresponding sequences of the model strains of each genus were selected for download (Yoon et al. 2017). Multiple sequence alignment was performed using MEGA and Genedoc to identify conserved regions within the genus Veillonella, and then, these conserved regions were compared with the gene sequences of other genera to obtain intergenera-specific loci and regions, which were recorded as SNP loci and high specificity regions.

Primer and probe design

The 16S rRNA gene sequences were used for the primer and probe design. According to the principles of primer and probe design, a pair of primers was designed in the highly conserved region, and the specificity of primer and probe were analyzed using NCBI Primer-BLAST, and Primer Primier 5 was used to predict the formation of primer dimer and secondary structure. The primer and probe were synthesized and purified using high-performance liquid chromatography (Sangon Biotech, Shanghai, China).

Construction of recombinant plasmids

The conserved sequence of V. parvula 16S rRNA gene (431–567) was amplified using the designed primers according to the manufacturer’s instructions, and the PCR amplification product was cloned into vector pGM-T (Tiangen Biotech, Bei**g, China). The plasmids were detected using a NanoDrop spectrophotometer. Then, standard recombinant plasmids were prepared at 10-fold dilutions ranging from 108 to 100 copies/μL for sensitivity analysis. Plasmid concentration and copy number were determined using the formula: DNA copy number (copies/μL) = [6.02 × 1023 × plasmid concentration (ng/μL) × 10−9]/[DNA length (nucleotides) × 660]. The length of DNA (3152 bp) was the sum of plasmid fragment length (137 bp) and vector length (3015 bp).

Real-time quantitative PCR assay

The qPCR assay was performed in a reaction volume of 20 μL using the premixed reaction solution TIANGEN SuperReal PreMix (TIANGEN, China). PCR conditions were as follows: 10 μL Supermix for probes, 0.6 μL upstream primer, 0.6 μL downstream primer, 0.4 μL probe, 6.4 μL ddH2O, 2 μL DNA; temperature cycling: predenaturing at 95 °C for 15 min, 40 cycles of PCR at 95 °C for 3 s and at 60 °C for 30 s.

Droplet digital PCR assay

The ddPCR assay was performed in a reaction volume of 20 μL using a commercial ddPCR kit (**nyi, Bei**g, China). PCR mixture containing 15 μL Supermix for probes, 2.4 μL upstream primer, 2.4 μL downstream primer, 0.75 μL probe, 8.45 μL ddH2O, and 1 μL DNA. Twenty microliters of PCR mixture and 180 μL Droplet generation oil were mixed, and droplet generation was performed using the test instrument Drop Maker M1. The droplet emulsion was thermally cycled in the following conditions: predenaturing at 95 °C for 10 min, 40 cycles of PCR at 94 °C for 30 s, and at 60 °C for 1 min, and the test instrument was cooled at 12 °C for 5 min, and then, the reaction was finished. The completed reaction was removed from the small centrifuge tube, put into the microdrop detection chip. Of droplet detection oil, 430 μL and 500 μL were added into the oil wells and put into the Chip Reader Biochip Analyzer for analysis. ChipReaderR1 software was used to determine the nucleic acid copy number.

Simulation of sample preparation

After V. parvula were incubated for 48 h under anaerobic conditions at 37 °C in BHI, the bacterial solution was 10-fold diluted with BHI and spread it on Columbia agar containing 2% sodium lactate. After overnight anaerobic culture, observe the growth of V. parvula in Columbia agar, count the colony formation units, and dilute the bacterial solution according to the results. One milliliter of the V. parvula solution was diluted to the appropriate concentration, and genomic DNA was extracted according to the guidelines of TIANamp Bacteria DNA Kit (TIANGEN, China), and qPCR and ddPCR were performed after gradient dilution. Another 1 mL of V. parvula solution was diluted to the appropriate concentration and added to 200 mg of inactivated stool. Inactivated feces were sterilized using an autoclave at 121 °C for 30 min. Fecal genomic DNA was extracted with reference to the TIANamp stool DNA kit (TIANGEN, China), and gradient dilution was used for qPCR and ddPCR assay.

Assessing clinical testing capabilities of assay systems using isolation methods

A stool sample from a patient with IBD was used for the detection of Veillonella. Of the sample, 200 mg was accurately weighed for DNA extraction, and the abundance of Veillonella in the sample was calculated by qPCR. A gradient dilution of 1 g of stool sample was applied to OVSA medium. For each liter of OVSA medium containing 40 g TSA, 21 mL sodium lactate, 50 mL sheep blood, 16 mg vancomycin, and 1 mg oxacillin sodium, sheep blood and antibiotics were added after the basal medium was sterilized and cooled to 50 °C. The sample was incubated anaerobically for 48 h. A monoclonal colony was selected for thermal lysis and the lysed DNA was used for PCR amplification. The amplified samples were sent to Biotech Bioengineering (Shanghai, China) for sequencing. The ability of the assay system to detect clinical samples was evaluated by isolation and enumeration of Veillonella.

Results

Multiple sequence alignment and primer design

The full-length 16S rRNA gene sequences of 30 genera were aligned and five single nucleotide polymorphisms (SNPs) (at positions 452, 1026, 1037, 1142, and 1172) were found to be conserved among 15 species of Veillonella but specific to other genera of the Veillonellaceae and other common intestinal genera. Furthermore, three highly specific regions were found (at positions 171–173, 503–505, and 1017–1019), which could be used as alternative sites for primer and probe design (Fig. S1; Table 2).

Table 2 Interspecific loci of Veillonella spp.
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After evaluation, it was found that SNP sites 452 and 1172, and high-specificity regions 503–505 and 1017–1019, were of sufficient length to constitute a conserved Veillonella sequence. According to the guidelines for primer and probe design (provide by TargetingOne Corporation, Bei**g, China), an amplified fragment should be less than 150 bp. Site 1172 was located too far from the high-specificity region 1017–1019 to satisfy this condition. Instead, SNP 452 was selected as the 3' end of the upstream primer, high-specificity region 503–505 was selected as the central region of the probe, and the conserved sequence 549–567 was selected as the downstream primer. We thereby obtained a pair of primers and a probe with both intra-genus conservation and inter-genus specificity for Veillonella (Fig. 1). The Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) predicted no cross-reactivity with other species and high specificity. A pair of primers and a probe were used for subsequent analysis (Table 3).

Fig. 1
figure 1

The primer and probe sequences of Veillonella. Asterisk denotes the site that determines the multiple subtypes of Veillonella species (i.e., A for V. montpellierensis, G for the other 14 species)

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Table 3 Primer sequences used in this study
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Evaluation of the specificity and sensitivity of the qPCR assay

Four species of Veillonella and seven clinically common intestinal pathogens, E. faecalis, K. pneumoniae, M. morganii, E. coli, P. mirabilis, S. enteritidis, and S. dysenteriae, were detected to verify the specificity of the qPCR test. The results showed that the amplification curves of Veillonella and other strains could be clearly distinguished. The Ct value of H2O was 36.2 when a fluorescence value of 1000 was used as the baseline, which indicated that some environmental factors interfered with the identification of Veillonella (e.g., aerosols containing Veillonella DNA). To fully exclude environmental factors from interfering with the identification of Veillonella, Ct = 35 was chosen to distinguish between positive and negative fluorescence signals. Using this threshold, only V. dispar, V. nakazawae, V. parvula, and V. rodentium produced positive fluorescent signals, while the other species tested produced negative fluorescent signals (Fig. 2a).

Fig. 2
figure 2

The sensitivity and specificity of the qPCR assay. a The specificity of qPCR. b The sensitivity of qPCR. c Standard curve for qPCR sensitivity detection. The results of qPCR are shown by an amplification curve, and the legend is arranged according to the Ct values from smallest to largest. The dotted line is the fluorescence threshold

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The qPCR sensitivity was tested using standard plasmids. The concentration of plasmid template determined by the Nanodrop spectrophotometer was 11.0 ng/μL, the conversion copy number was 3.234 × 109 copies/μL, and the concentration of recombinant plasmid at a 10-fold serial dilution was 108–100 copies/μL. The results of sensitivity analysis are shown in Fig. 2b. The Ct value was 11.99 when the plasmid concentration was 108 copies/μL and 33.98 when the plasmid concentration was 102 copies/μL. A concentration range from 108 to 102 copies/μL conformed to linearity with an R2 value of 0.9994 (Fig. 2c). Furthermore, the Ct value was 35.56 when the plasmid concentration was 101 copies/μL, which exceeded the specificity test determined threshold and was not represented on the standard curve; therefore, the lower limit of qPCR detection was determined to be 100 copies/μL.

Evaluation of the specificity and sensitivity of the ddPCR assay

To verify the specificity of the ddPCR assay, the assay was conducted to detect V. parvula, V. nakazawae, and the common clinical pathogens, K. pneumoniae, S. enteritidis, P. mirabilis, E. faecalis, and E. coli. Among them, V. parvula and V. nakazawae produced high intensity positive fluorescent signals with copy numbers of 153,614.4 copies/μL and 170,915.1 copies/μL, respectively, while all other strains tested produced lower intensity fluorescent signals of 1.3 copies/μL for K. pneumoniae, 2.4 copies/μL for S. enteritidis, 0 copies/μL for P. mirabilis, and 3.5 copies/μL for E. faecalis, and the copy numbers of all other test organisms were fewer than 5 copies/μL (Fig. 3a). The number of droplets generated for each sample exceeded 30,000 microdroplets, and the threshold amplification number of greater than 3000 basically excluded the interference of other genera and environmental factors on the identification of Veillonella.

Fig. 3
figure 3

The sensitivity and specificity of the ddPCR assay. a The specificity of ddPCR. b The sensitivity of ddPCR. The results of ddPCR are shown by an FAM scatter graph. c Standard curve for ddPCR sensitivity detection

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The same plasmid template as that used for qPCR was then applied to test the sensitivity of ddPCR. The plasmid copy number was 3.234 × 109 copies/μL, and the concentration of recombinant plasmid at 10-fold serial dilutions was 106–100 copies/μL. The ddPCR method was used to detect the Veillonella 16S rRNA gene, and the results of sensitivity analysis are shown in Fig. 3b. When the plasmid concentration was greater than 104 copies/μL, which was beyond the detection range of ddPCR, the copy number data were not quantitatively significant. The copy number was 11,055 copies/μL at a plasmid concentration of 104 copies/μL and 11.3 copies/μL at a plasmid concentration of 101 copies/μL. The log-linear R2 of the copy number results was 0.9986 at plasmid concentrations of 104–101 copies/μL, and the copy number was detected when the plasmid concentration was 101 copies/μL (Fig. 3c). When the plasmid concentration was 100 copy/μL, the detected copy number was 0 copy/μL, which was lower than the negative control and was not on the standard curve, so the lower limit of ddPCR detection was determined to be 11.3 copies/μL.

Comparison of the detection capabilities of qPCR and ddPCR

The number of bacterial colonies of 100μL V. parvula grown by overnight anaerobic culture on Columbia agar plates was 107 CFU, corresponding to a bacterial concentration in solution of 108 CFU/mL. Based on these results, the extracted genomic DNA of V. parvula was diluted across a gradient and assayed using qPCR and ddPCR to compare the ability of the two methods to detect V. parvula in the bacterial solution. The results of qPCR are shown in Fig. 4a. The Ct values were linear with an R2 value of 0.9960 at concentrations ranging from 108 to 102 CFU/mL. It was therefore concluded that qPCR was more accurate for the detection of 102–108 CFU/mL of Veillonella in a bacterial solution (Fig. 4b). The results of ddPCR are shown in Fig. 4c. The logarithm of the corresponding copy number results was linear with an R2 value of 0.9912 when the concentration ranged from 104 to 101 CFU/mL. It was therefore concluded that ddPCR was more accurate for the detection of 101–104 CFU/mL of Veillonella in a bacterial solution (Fig. 4d).

Fig. 4
figure 4

Comparison of the detection capacities of qPCR and ddPCR for Veillonella. a The sensitivity of qPCR. b Standard curve for qPCR sensitivity detection. c The sensitivity of ddPCR. d Standard curve for ddPCR sensitivity detection

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To further investigate the ability of qPCR and ddPCR to detect Veillonella in samples, we simulated clinical samples by adding bacterial suspension to inactivated stool samples. The extracted fecal genomic DNA corresponded to a concentration of 108 CFU/mL of V. parvula. qPCR and ddPCR were performed after gradient dilution of fecal genomic DNA to compare the detection abilities of the two methods using the simulated samples. The results of the qPCR assay are shown in Fig. 5a. The corresponding Ct values were linear with an R2 value of 0.9972 at concentrations ranging from 108 to 103 CFU/mL. It was therefore concluded that qPCR was more accurate for the detection of 103–108 CFU/mL of Veillonella in the samples (Fig. 5b). The results of ddPCR are shown in Fig. 5c. The logarithm of the corresponding copy number results was linear with an R2 value of 0.9944 at concentrations ranging from 104 to 101 CFU/mL. It was therefore concluded that ddPCR was more accurate for the detection of 101–104 CFU/mL of Veillonella in fecal samples (Fig. 5d).

Fig. 5
figure 5

Comparison of the detection capacities of qPCR and ddPCR for Veillonella in simulated samples. a The sensitivity of qPCR. b Standard curve for qPCR sensitivity detection. c The sensitivity of ddPCR. d Standard curve for ddPCR sensitivity detection

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Evaluation of the clinical testing capability of the assay system by isolation and culture

The DNA concentration of a 200 mg clinical sample, as determined by the Nanodrop assay, was 98.3 ng/μL, and the Ct value was 17.86 (Fig. 6a) using qPCR to detect Veillonella, and the abundance was calculated to be 8.04 × 106 CFU/mL (Fig. 6b) according to the standard curve. Then, the content of Veillonella in 1 g of stool sample was found to be 4.02 × 107 CFU/mL, and the sample was diluted across a gradient according to qPCR and ddPCR. Then, 100 μL of each dilution (103–106) was speaded on Veillonella selection medium(OVSA), and a bacterial count of 3.9 × 107 CFU/mL was obtained after anaerobic incubation for 48 h, which was similar to the molecular detection result (Fig. 6c). Single clone was selected for sequencing, and sequencing data showed that V. dispar, V. nakazawae, and V. rodentium were identified.

Fig. 6
figure 6

Abundance testing of Veillonella spp. in clinical samples. a qPCR results, asterisk indicates samples. b The abundance of Veillonella was calculated from the qPCR results using the standard curve. c Spread plate to detect the abundance of Veillonella in the sample. Veillonella colonies are white and opaque and are boxed in yellow in the figure

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Discussion

An abnormal abundance of bacteria is closely associated with human disease. Advances in sequencing technology have revealed perturbations in the populations of an increasing number of anaerobic bacteria, and the potential link with disease has attracted the attention of researchers. As a class of opportunistic pathogen, Veillonella has been reported to be potentially associated with numerous diseases. Because of the stringent culture conditions and long incubation time required for Veillonella growth, the detection of Veillonella in patient samples by molecular methods would significantly reduce the diagnosis time. Previous studies have mainly focused on detection of Veillonella at the species level. For example, Rôças et al. reported the accurate detection of V. parvula by PCR (Rôças and Siqueira Jr 2006), Izumi et al. reported the accurate detection of V. tobetsuensis (Mashima and Nakazawa 2013) and V. infantium (Mashima et al. 2018a) by DnaK gene detection, and Igarashi et al. (Igarashi et al. 2009) and Mashima et al. (Mashima et al. 2016) accurately detected a variety of oral Veillonella species based on the rpoB gene. Detection of Veillonella at the species level is essential because of the distinct roles played by different Veillonella in disease. However, in the testing of clinical samples with no clear source of infection, species-level testing has limitations in terms of its narrow detection spectrum and high workload. In addition, only 15 species of Veillonella have been reported to date, but unknown species of Veillonella are still to be explored by amplicon sequencing, which could be overlooked when using species-level methods. The establishment of a quantitative method for the detection of Veillonella at the genus level not only allows for efficient and rapid determination of the extent of Veillonella infection in patients and therefore enables effective and timely treatment, but also provides the potential for detecting more undefined Veillonella species. In previous studies, researchers have generally neglected the quantification and detection of Veillonella at the genus level. Therefore, new detection methods for Veillonella at the genus level were needed.

In this study, we developed a method to quantify the 16S rRNA genes of 15 species of Veillonella using qPCR and ddPCR, which offered advantages in terms of detection range and accuracy compared with previous reports. The results of sample testing using the two assays showed that qPCR was more accurate for the detection of 103–108 CFU/mL of Veillonella in samples, and ddPCR was more accurate for the detection of 101–104 CFU/mL of Veillonella in samples. This indicated that ddPCR was more suitable for the detection of Veillonella in low-abundance samples with higher detection sensitivity, and high-concentration samples could be further diluted until the detection range is achieved. By contrast, because Veillonella commonly colonizes the human oral cavity, qPCR for Veillonella is more suitable for clinical applications and can be used to supplement the quantification of Veillonella with ddPCR when a sample has a low Veillonella content that cannot be quantified by qPCR. The combination of these two methods provides accurate quantification of Veillonella in samples at concentrations ranging from 101 to 108 CFU/mL.

IBD is a group of nonspecific chronic inflammatory disease of the gastrointestinal tract in which the microbiota of the gut of affected patients differs significantly from that of healthy individuals and is accompanied by elevated nitrate levels and a decreased concentration of short-chain fatty acids (Schirmer et al. 2019). As a class of anaerobic bacteria that use nitrate as a redox receptor, elevated nitrate levels promote the increased abundance of Veillonella (Rojas-Tapias et al. 2022). During the course of our study, a stool specimen from a patient with IBD was collected and subjected to conventional smear and molecular assays. The results showed that the detected abundance of Veillonella was essentially the same between the two methods at 3.9 × 107 CFU/mL and 4.02 × 107 CFU/mL, respectively. The accuracy of the assay system was verified by the gold standard method of spread plate.

In this study, we isolated V. dispar, V. nakazawae, V. rodentium, and other species of Veillonella from clinical samples of patients with IBD using our newly developed assay system. The qPCR assay can rapidly detect both DNA and single colonies directly following thermal lysis, with accurate results and without the need for electrophoresis. Currently, this detection system can accurately detect Veillonella at the genus level but cannot further identify the corresponding species. In subsequent studies, we will combine it with molecular detection at the species level to obtain more detailed results. During laboratory testing, environmental factors, such as aerosols, can interfere with the experimental results, and negative pair setting is necessary to determine the threshold for a positive result to ensure accuracy.

In summary, the present study was conducted to establish a quantitative detection system for Veillonella spp. in simulated samples. This developed assay may be applied clinically to detect changes in Veillonella spp. in patients in a timely manner, as well as for research purposes to potentially aid the discovery of new species of Veillonella.