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Polymerase chain reaction primers for quantitatively detecting Ochrobactrum anthropi, kit and method for quantitatively detecting Ochrobactrum anthropi

A paleobacterial, quantitative detection technology, applied in microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of errors, high cost, slow speed, etc. Detecting fast effects

Pending Publication Date: 2020-06-09
威海以琳生物科技有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the judgment method based on ribosomal RNA gene sometimes makes mistakes, because some strains have very different taxonomy but have highly similar ribosomal RNA gene fragments; BIOLOG method is also used to detect Pallidum pallidus, but the speed is slower Slow, and the corresponding cost is relatively high (J Environ Sci(China).2009; 21(10):1446-51.Isolation of marine benzo[a]pyrene-degrading Ochrobactrumsp.BAP5 andproteins characterization)

Method used

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  • Polymerase chain reaction primers for quantitatively detecting Ochrobactrum anthropi, kit and method for quantitatively detecting Ochrobactrum anthropi
  • Polymerase chain reaction primers for quantitatively detecting Ochrobactrum anthropi, kit and method for quantitatively detecting Ochrobactrum anthropi
  • Polymerase chain reaction primers for quantitatively detecting Ochrobactrum anthropi, kit and method for quantitatively detecting Ochrobactrum anthropi

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the verification of paleobacterium microorganism-specific primer pair

[0045] The designed primer pair OA3F-OA3R was used for PCR amplification experiment. The PCR reaction system (12 μL) was: NPK02buffer (2×) (Weihai Xiaodong Biology) 6 μL, species-specific primers 1.5 μL (including both upstream and downstream primers, concentration 2 μM), template 1 μL (metagenomic DNA extracted from pit mud soil samples and marine biofilm samples as template DNA), Taq enzyme 0.25 μL, dd H 2 O 3.25 μL.

[0046] PCR reaction cycle parameters: 94°C for 3min; 94°C for 30s, 60°C for 30s, 72°C for 1min, 37 cycles; 72°C for 2min.

[0047] Agarose gel electrophoresis: 1% agarose gel, 4 μL of MarkerB sample, 12 μL of amplification product + 3 μL of loading buffer, and an appropriate amount of ethidium bromide (10% EB) was added to the buffer. The voltage is 110V, the current is above 40mA, observe once every 20min, and stop electrophoresis for about 40min.

[0048] The spec...

Embodiment 2

[0063] The specificity of the DNA fragment detected in embodiment 2 in other pale bacilli

[0064] The OA3F-OA3R primers were used to amplify the sequence of Paleobacterium pallidum in the rice field sludge metagenome, and the PCR product was sequenced using OA3R unidirectionally, and the sequencing results were converted into reverse complementary sequences (forward sequence), the results are as follows:

[0065] >1

[0066] AACGGGAACCCGTCGAAGAACGAGCCGCCGGCGAACAGCACGATCAGCAGGATGGCGAACAGGAAGCTCGGAATGGCGTAGCCGACGATGATGACGGCGCTGGTCCAGGTGTCGAAGCGCGAGCCGTCCTTGATCGCCTTGCGGATGCCGAGCGGGATCGAGATCAGGTAAGGGACAGCAAGGTCGAACCAAGGCCCGAAC 201 bases

[0067] Expected sequence:

[0068] >2

[0069] CAACGGGAACCAGTCGAAGAACGAGCCGCCCGCAAACAGTACGATCAGCAGGATTGCAAACAGGAAGCTTGGAATGGCGTAGCCGACGATGATGACGGCGCTGGTCCAGGTGTCGAAGCGCGAACCGTCCTTGATTGCCTTGCGAATGCCGAGTGGAATCGAGATCATGTAGGACAGAAAAGTCAGCCACAGGCCGAGCGAGATTGAAACCGGCATC 217碱基(Ochrobactrum anthropi strain OAB chromosome 1,Range:263890to 264106)(Ochro...

Embodiment 3

[0073] Example 3 Using OA3F-OA3R Primers to Quantitatively Determinate the Content of Paddy Paddy Soil Samples

[0074] In this embodiment, the compound bacteria preparation contains Pediococcus acidilactici, Lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus plantarum, Lactobacillus pentosus lactobacillus pentosus, Lactobacillus paracasei Lactobacillus paracasei, Agrobacterium tumefaciens, Serrat Serratia sp.JL, Lactobacillus fermentum, Agrobacterium Rhizobium larrymoorei, iron-based autotrophic nitrifying bacteria Microbacterium testaceum, Luteimonas cucumeris, Ochrobactrum sp.JL, Lactobacillus casei, nasturtium Fructobacillus tropaeoli, Acetobacter setunensis.

[0075]QPCR determination of Pallidum pallidum in a series of paddy field soil samples: Two soil samples with and without complex bacterial preparations were extracted by conventional methods. Use the Ezup column genome extraction kit (B518251, Shanghai Sangong) to extract 32 samples (including 16 gener...

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Abstract

The invention provides a pair of primers for detecting Ochrobactrum anthropi. The primers can rapidly determine any flora samples containing the Ochrobactrum anthropi so as to realize the specific recognition on the Ochrobactrum anthropi. A provided method includes extracting the metagenome of a to-be-detected sample, and performing dilution and split charging; respectively taking a small amount of the diluted metagenome sample to equally mix, and using a pair of primers to perform amplification; taking an amplified product as a QPCR quantitative standard stock solution through concentration determination; and performing the quantitative determination of the Ochrobactrum anthropi on all samples. The interference from other strains with highly similar ribosomal RNA gene fragments can be avoided by adopting the provided primers to detect the Ochrobactrum anthropi; and therefore, accurate determination on the Ochrobactrum anthropi can be realized.

Description

technical field [0001] The application relates to the field of biological detection and microbiological technology, in particular to a polymerase chain reaction primer, a kit and a method for quantitatively detecting Pallidum bacillus. Background technique [0002] Pallidum is a bacillus with parallel sides and rounded ends, usually solitary; cells stain Gram-negative; motility via periphyte flagella; obligate aerobic, strictly respiratory metabolism, with oxygen as the terminal electron acceptor , can use various amino acids, organic acids and carbohydrates as carbon sources; the optimum growth temperature is 20-37°C; the colonies on nutrient agar are colorless; in a low temperature environment, a variety of Cellulase is used to decompose crops such as corn stalks, thereby improving soil physical and chemical properties and improving soil fertility. The emulsification index of the Paleobacterium pallidum fermentation broth is relatively high, which has a strong emulsificat...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2565/125
Inventor 黄婕吉鸿睿金龙史宏伟张治洲
Owner 威海以琳生物科技有限责任公司
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