DSMG-Chip: A High-Throughput Degenerate qPCR Chip for Profiling Microbial DMSP and Related Organic Sulfur Metabolic Genes in Diverse Environments

The organosulfur cycle involves active microbial transformations of dimethylsulfoniopropionate (DMSP) to yield the climate active gas dimethyl sulfide (DMS) and other compounds. The lack of rapid and accurate methods to quantify the DMSP metabolic potential hinders a deeper understanding of this cycling. We developed a high-throughput qPCR (HT-qPCR) chip, DSMG-chip, to quantify the absolute abundance of DMSP and related organic sulfur metabolic genes. DSMG-chip contains 42 degenerate primer sets targeting 27 organosulfur metabolic genes, with the 16S rRNA gene as a reference, allowing for the detection of 41 environmental samples simultaneously. In silico analysis indicated that the DSMG-chip possesses broad taxonomic coverage (1.4–91.3%, spanning 12 phyla and 275 genera) and high specificity (44.4–100%, mean: 85.34%). Validation experiments using conventional PCR, qPCR, and HT-qPCR confirmed the primers’ strict specificity, robust amplification efficiency (0.677 to 0.997, mean: 0.771), and excellent accuracy, correlating well with conventional qPCR (Pearson’s r = 0.914). Finally, application of the DSMG-chip accurately reflected variations in DMSP metabolism across diverse seawater and sediment samples, highlighting the active microbial DMSP cycling in Pacific Ocean (section P) seawater and the contribution of the methanethiol (MeSH) methylation pathway (via mddA) in sediments. Our findings provide a powerful, novel tool for studying the organosulfur cycle.有机硫循环的核心环节是二甲基巯基丙酸内盐（dimethylsulfoniopropionate, DMSP）的活性微生物转化过程，该反应可生成具有气候活性的气体二甲基硫（dimethyl sulfide, DMS）与其他代谢产物。目前缺乏可快速精准定量二甲基巯基丙酸内盐代谢潜能的方法，这一短板阻碍了学界对该硫循环过程的深入认知。本研究开发了一款高通量定量PCR（high-throughput quantitative PCR, HT-qPCR）芯片DSMG-chip，用于定量二甲基巯基丙酸内盐及相关有机硫代谢基因的绝对丰度。该芯片包含42条简并引物组，靶向覆盖27个有机硫代谢基因，并以16S rRNA基因作为内参，可同时完成41份环境样本的检测。虚拟仿真分析结果显示，DSMG-chip具有广泛的分类学覆盖范围（1.4%~91.3%，覆盖12个门、275个属），且特异性优异（44.4%~100%，均值为85.34%）。通过常规PCR、定量PCR及高通量定量PCR开展的验证实验证实，该芯片引物具有严格的特异性、稳定的扩增效率（0.677~0.997，均值为0.771）及优异的定量准确性，其结果与常规定量PCR呈现极强的相关性（Pearson相关系数r=0.914）。最后，将DSMG-chip应用于实际样本检测，可精准反映不同海水与沉积物样本中二甲基巯基丙酸内盐代谢的差异；该分析结果揭示了太平洋P断面海水中活跃的微生物介导的二甲基巯基丙酸内盐循环过程，以及沉积物中甲基硫醇（methanethiol, MeSH）甲基化通路（通过mddA基因）的贡献。本研究成果为有机硫循环的相关研究提供了一款高效且新颖的研究工具。