A microbial source tracer molecular marker and its high-throughput detection method for the detection of various fecal pollution sources

Abstract

The invention discloses a microbial source tracking molecular marker and a high-throughput detection method thereof for detecting multiple fecal pollution sources. The molecular marker is composed of upstream and downstream primers and probes; sequences of the upstream and downstream primers and the probes are shown as SEQ ID NO:1-74, wherein a pair of upstream and downstream primers are shown as SEQ ID NO:1,2, and the corresponding probe is shown as SEQ ID NO:3; a pair of upstream and downstream primers are shown as SEQ ID NO:4,5, and the corresponding probe is shown as SEQ ID NO:6; by such analogy, a pair of upstream and downstream primers are shown as SEQ ID NO:72,73, and the corresponding probe is shown as SEQ ID NO:74; and the 5' and 3' terminals of all probes are connected to fluorescent reporter groups and fluorescent quencher groups. The molecular marker and the detection method provided by the invention can be used for conducting 5184 quantitative PCR reactions in one time and can be used for rapidly and effectively detecting fecal pollution of 216 samples simultaneously, so that the shortcoming of original means which is low in throughput is overcome; and the molecular marker and the detection method are high in sensitivity and specificity.

Description

technical field [0001] The invention relates to biotechnology, in particular to a microbial source tracer molecular marker for detection of various feces pollution sources and a high-throughput detection method thereof. Background technique [0002] Increasing water pollution has posed a major threat to human survival and has become a major obstacle to human health, economic and social sustainable development. Microbial pollution of water bodies (including parasites, viruses, and pathogenic bacteria) can cause a variety of infectious and parasitic diseases, posing an important threat to water quality safety and human health. Fecal pollution is one of the main sources of microbial pollution in water bodies. The host feces of different species have different degrees of harm to water pollution due to the differences in their intestinal microorganisms. Distinguishing and identifying the source and degree of fecal pollution in water bodies will help water microorganisms The supe...

AI Technical Summary

Problems solved by technology

[0004] Although gratifying progress has been made in the use of TaqMan probe quantitative PCR method for microbial source tracing, due to technical and workload limitations, previous studies often only focus on one or a few molecular markers. The detection of fecal pollution sources, which underestimates the possible cross-reaction between many undetected host feces and molecular markers, and environmental samples usually have the characteristics of compound pollution, so it is difficult to accurately trace the source of multiple fecal contamination; Secondly, studies have shown that fecal host intestinal microbes have obvious geographical distribution and are affected by diet structure, which makes the use of host-specific molecular markers have certain geographical limitations, and can be used in certain areas with high sensitivity and specificity. Molecular markers cannot be well applied in other areas, so it is necessary to use multiple molecular markers to improve the accuracy of the detection results of fecal pollution sources; finally, the traceability of fecal pollution usually requires the detection of a large number of samples in a certain area. Due to the limitation of throughput, it is difficult to realize the rapid detection of various molecular markers and a large number of samples by the fluorescent quantitative PCR method.

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