Primer for estimating methane output from rumen

Abstract

To provide means for fast and simple quantification of bacteria that inhibit methane production.SOLUTION: The present invention provides a primer set targeting uncultured bacteria of the Succinivibrionaceae family for detection, and a primer set targeting Prevotella lacticifex for detection.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel primer and a method for estimating methane production in the rumen using the primer, and more particularly to a method for estimating methane production in the rumen using the primer. [Background technology]

[0002] Ruminant livestock break down and ferment the feed they ingest, using the microorganisms that live in their first stomach, and use the short-chain fatty acids that are the product of fermentation as a source of nutrition. Meanwhile, hydrogen produced during the process of producing short-chain fatty acids is converted into methane and released into the atmosphere. There are 1.5 billion cattle raised in the world, and methane derived from fermentation in the digestive tracts of ruminant livestock is a major source of greenhouse gas emissions, accounting for 40% of greenhouse gas emissions from the agricultural sector. Efforts to reduce greenhouse gas emissions are being made all over the world, and research and development into technologies to reduce cattle-derived methane is being carried out in various countries.

[0003] In the development of methane reduction technology, research is also being conducted focusing on individual differences in cattle. In other words, efforts are being made to clarify the microbial characteristics of individual cattle that naturally produce little methane, and to utilize these characteristics in the form of probiotics, etc., or to link them to the breeding of low-methane-producing cattle. It is known that a characteristic of low-methane-producing cattle is an increase in propionic acid production, which is in an antagonistic relationship with methane production over hydrogen consumption in the cattle rumen. It has been pointed out that uncultured bacteria such as Succinivibrionaceae are associated with low-methane-producing cattle, but they have not yet been cultured. In addition, a new bacterial species, Prevotella lacticifex (hereinafter sometimes referred to as "P. lacticifex"), which was detected and isolated as a bacterium characteristic of low-methane-producing cattle specified by the National Agriculture and Food Research Organization, has been characterized as producing a large amount of a precursor of propionic acid, and it is expected that it will be used in applications to enhance propionic acid production and reduce methane production.

[0004] There are several methods for measuring methane emissions from cows, including 1) a chamber method in which the cow's entire body or head is covered to collect breath, 2) a method in which cows are given a trace amount of rare gas such as sulfur hexafluoride (SF6) as an internal standard and their breath is collected in a cylinder attached to their neck, and 3) a method for estimating methane from the methane:carbon dioxide ratio, but all of these require special equipment and facilities.In addition, individual management, such as measuring feed intake, may be required.

[0005] In order to detect microbial communities, uncultured Succinivibrionaceae bacteria and P. lacticifex and to investigate their proportion in the entire bacterial community, complex analytical procedures are required, such as PCR amplification of DNA extracted from the ruminal microbial community using universal primers (comprehensive primers targeting all bacteria) targeting partial sequences of the 16S rRNA gene (such as the V3-V4 region), deciphering the base sequences on a scale of tens of thousands of genes using a next-generation sequencer such as MiSeq, and analyzing them on a dedicated platform such as QIIME (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Caporaso, J., Lauber, C., Walters, W. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621-1624 (2012). Summary of the Invention [Problem to be solved by the invention]

[0007] Among the conventional techniques, methods for measuring gases require special devices and equipment, and may require the collection of data managed for each individual animal, such as measuring the amount of feed intake.

[0008] On the other hand, conventional detection of microbial groups is a research method for comprehensive occupancy surveys aimed at comparing samples, and does not provide quantitative data for specific bacteria. In addition, analysis at specialized testing institutions using partial sequences of the 16S rRNA gene (such as the V3-V4 region) cannot identify which sequences are uncultured Succinivibrionaceae bacteria or P. lacticifex. Specialized analysis is required for identification. In addition, samples must be sent to specialized research institutions, and analysis takes about a month.

[0009] As described above, both conventional methods for measuring methane and methods for estimating methane production from microbial communities have many problems. The present invention has been made under these circumstances, and aims to provide a means for quickly and simply quantifying bacteria that inhibit methane production. [Means for solving the problem]

[0010] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that PCR using primers consisting of the base sequences set forth in SEQ ID NOs: 5 to 8 can specifically amplify DNA of bacteria characteristic of low methane producing cows, and completed the present invention based on this finding.

[0011] That is, the present invention provides the following [1] to [5]. [1] A primer having any one of the following nucleotide sequences (a) to (d): (a) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO: 1; (b) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO: 2; (c) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO: 3; (d) A base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO:4.

[0012] [2] The primer according to [1], wherein the base sequences of (a), (b), (c), and (d) are base sequences of 15 or more and 25 or less bases.

[0013] [3] The primer according to [1], wherein the base sequences of (a), (b), (c), and (d) are the base sequences set forth in SEQ ID NOs: 5, 6, 7, and 8, respectively.

[0014] [4] The primer described in [1], which is used to estimate methane production from within the rumen.

[0015] [5] A method for estimating methane production from the rumen, comprising the following steps (1) to (4): (1) collecting ruminal fluid from a ruminant; (2) extracting DNA from the ruminal fluid collected in step (1); (3) performing quantitative PCR using the DNA extracted in step (2) as a template and the primers according to any one of [1] to [3], wherein the primers are a set of a primer having the base sequence of (a) and a primer having the base sequence of (b), and / or a set of a primer having the base sequence of (c) and a primer having the base sequence of (d); (4) A process for estimating the amount of methane production from within the rumen based on the results of the quantitative PCR in process (3). Effect of the Invention

[0016] The present invention provides a novel primer that can be used to rapidly and simply estimate methane production from the rumen. [Brief description of the drawings]

[0017]

Figure 1

[0018] The present invention will be described in detail below. (A) Primer The primer of the present invention is a primer having any one of the following base sequences (a) to (d): (a) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO:1 below (b) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO:2 below (c) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO:3 below (d) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO:4 below SEQ ID NO: 1: 5'-ACC GAT GAT C TC TAG CTG GTT TGA GAG GAC AA T CAG CCA CAT-3' SEQ ID NO: 2: 5'-CAC GGA GTT T GC CGG TGC TTA TTC ACA CTC T AA CGT CAA AT-3' SEQ ID NO: 3: 5'-GAG TAA CGC G TA TCC AAC CTG CCG CAA AG T AGG GCA CAG-3' SEQ ID NO: 4: 5'-GGG TAC CTG C AA TGA ATG ACA CGT CAC TC A CTT TAT CCC-3'

[0019] The primer consisting of the base sequence (a) is a forward primer targeting uncultured bacteria of the family Succinivibrionaceae, the primer consisting of the base sequence (b) is a reverse primer targeting uncultured bacteria of the family Succinivibrionaceae, the primer consisting of the base sequence (c) is a forward primer targeting Prevotella lacticifex, and the primer consisting of the base sequence (d) is a reverse primer targeting Prevotella lacticife.

[0020] In the base sequences (a) to (d), the number of consecutive bases may be 10 or more, preferably 12 or more, and more preferably 15 or more, and preferably 35 or less, more preferably 30 or less, and even more preferably 25 or less.

[0021] The primers consisting of the nucleotide sequences of (a), (b), (c), and (d) are preferably primers consisting of the nucleotide sequences set forth in SEQ ID NOs: 5, 6, 7, and 8, respectively (the underlined nucleotide sequences in SEQ ID NOs: 1 to 4 above). The primers consisting of the nucleotide sequences set forth in SEQ ID NOs: 5, 6, 7, and 8 are designed in consideration of not only the specificity of the nucleotide sequence, but also the site of use and the length of the sequence so as not to impair the function as a primer (for example, to prevent annealing between primers or self-annealing within the primer).

[0022] The primers of the present invention are targeted for detection of uncultured Succinivibrionaceae bacteria or Prevotella lacticifex. The number of these bacteria present in the rumen shows a positive correlation with the amount of propionic acid produced in the rumen. In addition, propionic acid production in the rumen is in an antagonistic relationship with methane production over hydrogen consumption. Therefore, it is considered that the number of uncultured Succinivibrionaceae bacteria and Prevotella lacticifex present in the rumen shows a negative correlation with methane production in the rumen. Therefore, the primers of the present invention can be used to estimate the amount of methane produced in the rumen. In addition, the primers of the present invention can also be used to estimate the amount of propionic acid produced in the rumen.

[0023] (B) Methods for estimating methane production The method of the present invention for estimating the amount of methane production in the rumen includes the following steps (1) to (4).

[0024] In step (1), ruminal fluid is collected from a ruminant.

[0025] There is no need to use a special method for collecting ruminal (first stomach) fluid, and it can be collected according to known methods. In addition, since dedicated equipment for collecting ruminal fluid is commercially available, it can be collected using such equipment.

[0026] The target ruminant is mainly cattle, but other ruminant animals such as goats, sheep, deer, etc. may also be targeted. The cattle may be dairy cows or beef cattle.

[0027] In step (2), DNA is extracted from the ruminal fluid collected in step (1).

[0028] Extraction of DNA from ruminal fluid does not require the use of any special method and can be carried out according to known methods. In addition, kits specifically for extracting DNA are commercially available, so the extraction can be carried out using such kits.

[0029] In step (3), quantitative PCR is carried out using the DNA extracted in step (2) as a template and the primers of the present invention.

[0030] When uncultured bacteria of the family Succinivibrionaceae are targeted for detection, a set of primers consisting of the base sequence (a) and the base sequence (b) is used, and when Prevotella lacticifex is targeted for detection, a set of primers consisting of the base sequence (c) and the base sequence (d) is used. When both bacteria are targeted for detection, the above two types of primer sets are used.

[0031] The type of quantitative PCR is not particularly limited, but real-time PCR is preferred. Real-time PCR can be performed by a known method, for example, SYBR TM Green, TaqMan TM This can be carried out according to a probe method using a probe or the like.

[0032] The denaturation conditions in PCR are not particularly limited, but the denaturation temperature is preferably 92 to 100°C, more preferably 94 to 98°C, and even more preferably about 95°C, and the denaturation time is preferably 5 to 180 seconds, more preferably 10 to 60 seconds, and even more preferably about 15 seconds.

[0033] The annealing conditions in PCR are not particularly limited, but the annealing temperature is preferably 45 to 68°C, more preferably 55 to 57°C, and even more preferably about 56°C, and the annealing time is preferably 10 to 60 seconds, more preferably 20 to 40 seconds, and even more preferably about 30 seconds.

[0034] The conditions for the extension reaction in PCR are not particularly limited, but the extension reaction temperature is preferably 60 to 75°C, more preferably 60 to 72°C, and even more preferably about 72°C, and the extension reaction time is preferably 10 to 60 seconds, more preferably 20 to 40 seconds, and even more preferably about 30 seconds.

[0035] The number of cycles in PCR is not particularly limited, but is preferably 20 to 60 cycles, more preferably 30 to 50 cycles, and further preferably about 40 cycles.

[0036] In step (4), the amount of methane production from within the rumen is estimated based on the results of the quantitative PCR in step (3).

[0037] The results of the quantitative PCR in step (3) show the number of uncultured Succinivibrionaceae bacteria or Prevotella lacticifex present in the rumen. As described above, the number of these bacteria present shows a negative correlation with methane production in the rumen. Therefore, the amount of methane produced in the rumen can be estimated from the results of the quantitative PCR in step (3). In addition, as shown in the examples described later, it is also possible to estimate the amount of methane produced in the rumen from the ratio of the number of target bacteria present divided by the total number of bacteria present in the rumen, rather than the number of bacteria present. The total number of bacteria present in the rumen can be determined, for example, by quantitative PCR using the primer set (SEQ ID NOs: 9 and 10) described in Table 1.

[0038] The method of the present invention makes it possible to quantify the amount of methane produced from the rumen of each individual ruminant, which makes it possible to identify individuals with low methane production and to use such individuals in breeding selection.

[0039] As described above, the number of uncultured Succinivibrionaceae bacteria or Prevotella lacticifex present in the rumen correlates with the amount of propionic acid produced in the rumen, so the method of the present invention can also be applied to a method for estimating the amount of propionic acid produced in the rumen. EXAMPLES

[0040] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0041] Materials and Methods [Design of specific primers] The 16S rRNA gene sequences of uncultured Succinivibrionaceae bacteria and Prevotella lacticifex were obtained from low methane-producing cows raised and managed at the Livestock Research Department of the National Agriculture and Food Research Organization by the following method. Ruminal fluid was orally collected from the low methane-producing cows using a ruminal fluid collection probe (Luminar, Sanshin Kogyo), and microbial DNA was extracted from the obtained ruminal fluid. The 16S rRNA gene was PCR amplified using the bacterial universal primer set 27f (5'-AGAGTTTGATCMTGGCTCAG-3') (SEQ ID NO: 11) and 1492r (5'-GGYTACCTTGTTACGACTT-3') (SEQ ID NO: 12), and an amplified fragment was obtained. Each amplified DNA fragment was introduced into pCR2.1 vector, and then introduced into E. coli for amplification. The nucleotide sequence of the plasmid obtained from the colony was decoded to obtain the nucleotide sequence of each 16S rRNA gene of uncultured Succinivibrionaceae bacteria and P. lacticifex. The nucleotide sequences were aligned using ClustalW, and sequence sites unique to each target bacterium were detected. In addition, the primer sequences were designed to avoid three-dimensional structure formation or self-annealing within the primer sequence and between the forward and reverse primers, and specific primers suitable for quantification were designed for each. The primer sequences were evaluated for their specificity by database searches at NCBI / EMBL / DDBJ. PCR was then performed on known bacterial DNA of closely related species of the primer target bacteria (Succinivibrio dextrinosolvens ATCC 19716, Succiniclasticum ruminis DSM 9236, Prevotella ruminicola ATCC 19189, Prevotella bryantii DSM 11371, Prevotella brevis ATCC 19188, and Prevotella albensis DSM 11370) using a clone plasmid of the primer target bacteria as a positive control, and the specificity of the primers was determined by the presence or absence of amplification.

[0042] [Correlation analysis between bacterial quantification and ruminal fermentation product indicators] From April 2017 to November 2021, ruminal fluid samples were orally collected three hours after early morning feeding from a total of 71 Holstein lactating and dry dairy cows kept and managed at the National Agriculture and Food Research Organization's Livestock Research Department, and microbial DNA was extracted and purified from each sample (200 samples from lactating dairy cows and 24 samples from dry dairy cows).

[0043] The number of uncultured Succinivibrionaceae bacteria and P. lacticifex present in the rumen of each sample and the total number of bacteria in the rumen were quantified by real-time PCR. The primer sets used are shown in Table 1 (SEQ ID NOs: 5 to 10). [Table 1]

[0044] The total number of bacteria in the rumen was quantified using a previously reported primer set (primer set for total bacteria in the rumen in Table 1 (SEQ ID NOs: 9 and 10), Denman & McSweeney, 2006). TM SYBR TM The PCR was performed using Green qPCR SuperMix-UDG (Invitrogen). TM A 3 real-time PCR system (Applied Biosystems) was used. PCR conditions were 95°C for 15 seconds for thermal denaturation, 56°C for 10 seconds for annealing, and 72°C for 30 seconds for extension reaction, and 40 cycles were performed. Quantitative data was shown as the percentage of target bacteria divided by the total number of bacteria.

[0045] As an index of ruminal fermentation, the concentration of short-chain fatty acids in the rumen was measured using a gas chromatography short-chain fatty acid analysis system (8890 GC System (Agilent Technologies)). From the measured data, the short-chain fatty acid balance (acetic acid + butyric acid) / propionic acid was calculated, and the correlation with the bacterial quantitative data was analyzed.

[0046] 〔result〕 [Design of specific primers] The designed primer sequences were confirmed to be specific to the 16S rRNA genes of uncultured Succinivibrionaceae bacteria and P. lacticifex by database searches at NCBI, EMBL, and DDBJ. PCR confirmed that quantitative PCR was possible with both primer sets. Furthermore, PCR was negative when DNA from closely related bacterial species was used, demonstrating that each was a specific PCR quantitative system.

[0047] [Correlation analysis between bacterial quantification and ruminal fermentation product indicators] The abundance ratios of uncultured Succinivibrionaceae bacteria and P. lacticifex showed a negative correlation with (acetic acid + butyric acid) / propionic acid, and were higher in the high propionic acid producing rumen where (acetic acid + butyric acid) / propionic acid was 3.5 or less (Fig. 1). This indicates that the abundance ratios of these bacteria are positively correlated with propionic acid production in the rumen.

[0048] [References] 1. Denman SE & McSweeney CS (2006) Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol Ecol, 58(3):572-582. [Industrial Applicability]

[0049] The present invention can be used in industries such as the livestock industry.

Claims

1. A primer consisting of any one of the following nucleotide sequences (a) to (d): (a) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO: 1; (b) a base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO: 2; (c) a nucleotide sequence of 10 or more consecutive nucleotides contained in the nucleotide sequence set forth in SEQ ID NO: 3; (d) A base sequence of 10 or more consecutive bases contained in the base sequence set forth in SEQ ID NO:

4.

2. The primer according to claim 1, wherein the base sequences of (a), (b), (c), and (d) are 15 to 25 base sequences.

3. The primer according to claim 1, wherein the base sequences of (a), (b), (c), and (d) are the base sequences set forth in SEQ ID NOs: 5, 6, 7, and 8, respectively.

4. The primer according to claim 1, which is used to estimate methane production from within the rumen.

5. A method for estimating methane production from within the rumen, comprising the following steps (1) to (4): (1) collecting ruminal fluid from a ruminant; (2) extracting DNA from the ruminal fluid collected in step (1); (3) performing quantitative PCR using the DNA extracted in the step (2) as a template and the primers according to any one of claims 1 to 3, wherein the primers are a set of a primer having the base sequence of (a) and a primer having the base sequence of (b), and / or a set of a primer having the base sequence of (c) and a primer having the base sequence of (d); (4) A process for estimating the amount of methane production from within the rumen based on the results of the quantitative PCR in process (3).

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