Probe, combination probe, kit and detection method used for performing classification detection on ammonia oxidizing bacteria

Abstract

The invention discloses a probe, combination probe, kit and detection method used for performing classification detection on ammonia oxidizing bacteria. The probe has any one of nucleotide sequences as shown in SEQ ID NO.1-20, or a sequence obtained by modifying the skeleton of any one of the nucleotide sequences as shown in SEQ ID NO.1-20, wherein a modification site is at least one of A, C, G and T in the sequence. The invention further provides a direct RNA liquid-phase chip detection method, and the method can carry out mRNA direct detection on the ammonia oxidizing bacteria by utilizing aliquid-phase chip platform, and amplification steps including reverse transcription, polymerase chain reaction (PCR), cyclic shearing and the like are not needed. The probe provided by the inventionis an mRNA probe of an important functional gene amoA of the ammonia oxidizing bacteria, and can directly represent the denitrification activity of the ammonia oxidizing bacteria; the gene is only possessed by the ammonia oxidizing bacteria; and the specificity of the group of probes is stronger than that of an existing 16SrRNA molecular probe.

Description

Technical field[0001]The invention belongs to the technical field of genetic engineering, and specifically relates to a probe used for classifying and detecting ammonia oxidizing bacteria, a combined probe, a kit and a detection method.Background technique[0002]After the implementation of “source control and pollution reduction” and strict environmental law enforcement throughout the country, organic pollution in water bodies has been effectively curbed, and nitrogen and phosphorus pollution has become the main problem of water pollution. In most national standards, such as "Emission Standards for Pollutants from Urban Sewage Treatment Plants", ammonia nitrogen and total nitrogen are strictly controlled; and in many local standards, such as the "Water Pollution in Minjiang and Tuojiang Basins of Sichuan Province" The "Substance Emission Standard" also formulated more stringent ammonia nitrogen and total nitrogen emission standards. It can be seen that ammonia nitrogen pollution has ...

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Problems solved by technology

[0007] However, at present, there are few reported amoA gene probes, and only a few probes also have problems such as long sequences, many arrays, and low coverage.

The amoA gene probe reported by Bruns et al. is as long as 700 bases (BRUNS M A, FRIES M R, TIEDJE J M, et al. Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria [J]. Microb Ecol, 1998, 36 (3) :293-302.), easily lead to cross-hybridization and false negatives; the amoA gene probe reported by Ward et al. is as long as 70 bases, and the total coverage of 24 pairs of probes to the amoA gene in the current database (NCBI) is only 2.55% (WARD B B, EVEILLARD D, KIRSHTEIN J D, et al. Ammonia-oxidizing bacterial community composition in estuarine and °Ceanic environments assessed using a functional gene microarray [J]. EnvironMicrobiol, 2007, 9(10): 2522-38.), It is difficult to accurately reflect the abundance and activity of ammonia oxidizing bacteria in the sample; the amoA gene probe reported by Bouskill et al. is also 70 bases, and the total coverage of the 28 pairs of probes on the amoA gene in the database is only 2.7% (BOUSKILL N J, EVEILLARD D, O'MULLAN G, et al.Seasonal and annualre℃currence in betaproteobacterial ammonia-oxidizing bacterial populationstructure[J].Environ Microbiol,2011,13(4):872-86.); amoA reported by McTavish et al. The coverage of the gene probe for the amoA gene is less than 1% (MCTAVISH H, FUCHS J, HOOPER A. Sequence of the genecoding for ammonia monooxygenase in Nitrosomonas europaea[J]. J Bacteriol, 1993, 175(8): 2436-44. )

It can be seen that the existing probes have low coverage of the existing ammonia oxidizing bacterial community, and it is difficult to accurately characterize the ammonia oxidation abundance and activity of the sample. At the same time, the sequence is long, which easily leads to cross-hybridization and false negatives

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