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This study aimed to identify pregnancy-associated differentially methylated genes and develop an early pregnancy detection technology utilizing methylation-specific fluorescent probes. This study focused on differentially methylated regions in gene promoters and CpG islands to identify potential biomarkers for swine pregnancy. Initially， methylation-specific PCR （MSP） was employed for preliminary validation of these differentially methylated regions. Subsequently， target-specific fluorescent probes were designed to establish an early pregnancy detection method based on quantitative methylation-specific PCR （qMSP）， followed by methodological verification. Compared with empty pregnant sows， the blood of pregnant sows at 25-30 days of gestation showed high methylation of VBP1 and CpG_280， while CpG_1360 showed low methylation， indicating that these sites may be involved in pregnancy regulation. Using β-actin as an internal reference probe， methylation probes for VBP1， CpG_280， and CpG_1360 were designed to establish a qMSP detection method for preliminary application. Concordance analysis between this methylation probe-based detection and ultrasound （B-ultrasound） showed ROC curve AUC values of 0.713 8， 0.608 0， and 0.746 6 （all>0.5）， with qMSP concordance rates of 85.0%， 78.75%， and 85.0%， respectively. This study is the first to confirm that VBP1 and CpG_280 are hypermethylated while CpG_1360 is hypomethylated during early sow pregnancy. The fluorescence probe-based methylation detection technology targeting these genes offers a novel method for early pregnancy diagnosis in swine.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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