Development and application of a highly sensitive quadruple droplet digital PCR method for simultaneous quantification of sulfonamide resistance genes

Abstract

Sulfonamide resistance genes (sul genes) have a high detection rate and strong transmissibility. Therefore, there is an urgent need to develop more efficient detection methods to enhance the monitoring of sul genes. Current analytical methods are insufficient for the simultaneous and accurate quantification of all sulfonamides resistance genes. To overcome this limitation, a quadruple method was established by integrating droplet digital PCR (ddPCR) with the ratio-based probe-mixing strategy, achieving sensitive detection of sul1, sul2, sul3, and sul4 genes in diverse matrices. Correspondingly, the primers and probes of sul genes were meticulously designed and rigorously validated, and the critical parameters for ddPCR such as annealing temperature, concentrations of primers and probes were systematically optimized. As a results, the quadruple ddPCR method demonstrates excellent sensitivity with limits of detection (LOD) ranging from 3.98 to 6.16 copies/reaction, and good repeatability (coefficient of variation <25%), adequately meeting the requirement for accurate sul genes quantification. Furthermore, this new method was applied across 115 diverse samples, including human feces, animal-derived foods, sewage and surface water, achieving positive rates of 100% for sul1, 99.13% for sul2, 93.91% for sul3, and 68.70% for sul4, with sul genes concentration ranging from non-detection to 2.14 × 10⁹ copies/g. In summary, the developed quadruple ddPCR method has potential to serve as an efficient and sensitive tool for monitoring sul genes.

Keywords: animal-derived foods; droplet digital PCR; feces; quadruple detection; sewage; sulfonamide resistance genes; surface waters.

Figure 1

The principle and workflow of quadruple ddPCR. The FAM channel can detect signals of targets labeled with the FAM reporter group; the HEX channel can detect signals of targets labeled with the HEX reporter group. Negative droplets present as gray, the T1 and T4 labeled with FAM appear blue, the T2 and T3 labeled with HEX manifest green, and the droplets concurrently bearing FAM and HEX signals are orange.

Figure 2

Sampling point locations of surface water and wastewater treatment plant in Shijiazhuang. C, Cha River; H, Round-city Water System; M, Minxin River; S, Shijin River; T, Hutuo River; X, South Flood Discharge Channel.

Figure 3

Comparison of the outcomes of PCR, qPCR, and ddPCR in the detection of actual samples. Among the 20 samples, samples numbered 1 to 3 are positive for sul1, samples 3 to 6 are positive for sul2, samples 7 to 8 are positive for sul3, samples 9 to 11 are positive for sul4, and samples 12 to 20 are negative samples. (A) The amplification curve of qPCR. (B) The agarose gel electrophoresis graph of PCR. The 2 kb DNA marker (TIANGEN, China) are on both sides of the picture, and the PCR products of the actual samples are in the middle. Text in the figure records the name of sul genes and the expected length of the PCR product. (C) Detection results of sul1 by single-target ddPCR. (D) Detection results of sul2 by single-target ddPCR. (E) Detection results of sul4 by single-target ddPCR. (F) Detection results of sul3 by single-target ddPCR.

Figure 4

Optimize the triple ddPCR system. (A–C) Represent the outcomes of optimizing the annealing temperature. (D–F) Show that varying sul3 primer concentrations affects droplet cluster separation efficiency.

Figure 5

Optimize the quadruple ddPCR system. (A) Different probe concentration combinations of sul1 and sul4; H represents the probe with high concentration and L represents the probe with low concentration. (B) Different probe concentration combinations of sul2 and sul3, H represents the probe with high concentration, and L represents the probe with low concentration. (C) The 2D plot of optimized quadruple ddPCR.

Figure 6

Quantification linearity of quadruple ddPCR: the x-axis represents the concentration of serially diluted linearized plasmid solutions and the y-axis represents the detection results of quadruple PCR. The linear regression equation and the correlation coefficient (R²) are shown. (A) sul1 gene. (B) sul2 gene. (C) sul3 gene. (D) sul4 gene.

Figure 7

Logarithm of sul genes concentration in various samples. B1–B40 are human feces samples; F1–F20 are animal-derived food samples; W1–W20 are sewage samples; C1–C4 are surface water samples from Cha River; H1–H4 are surface water samples from Round-city Water System; M1–M13 are surface water samples from Minxin River; S1–S7 are surface water samples from Shijin River; T1–T4 are surface water samples from Hutuo River; X1–X4 are surface water samples from South Flood Discharge Channel. For each sample, the higher the concentration, the darker the color, and white indicates no detection.

Figure 8

The logarithm of the average concentration of sul genes across different samples: the x-axis represents the sample types, the y-axis represents the sul gene names, and the z-axis represents the logarithmic values of average concentrations, log10 (copies/g) or log10 (copies/mL).