JP5237576B2 - Method for detecting and identifying microorganisms with cyanide resolution - Google Patents

Description

  The present invention relates to a method for detecting and identifying microorganisms that play a role of detoxifying harmful substances in the fields of medicine and various industries (food, chemistry, etc.) and environmental conservation (biodegradation of pollutants and water treatment) at the gene level. .

  Bioremediation using microorganisms' ability to decompose cyanide has been studied as a technique for repairing (detoxifying) soil and groundwater contaminated with cyanide compounds (Patent Documents 1 and 2). When such a technique is applied to repair a polluted environment, the restoration status has been evaluated by analyzing the concentration of cyanide to be purified. However, this method alone cannot distinguish between the physicochemical reduction of cyanide and the effects of bioremediation, such as diffusion of cyanide from contaminated plumes and vaporization into highly toxic hydrogen cyanide gas. There wasn't. Therefore, it was necessary to show the basis for the degradation and detoxification of the cyanide in the environment by microorganisms by another method.

  In biological remediation techniques at oil-contaminated sites, methods have been used to measure the activity of microorganisms that purify petroleum components by measuring the total number of bacteria by microscopic observation and monitoring respiratory activity. Such a monitoring method is effective when there is a sufficient amount of organic substances derived from pollutants and it is possible to artificially increase the microbial activity in the soil environment artificially. On the other hand, the microbial purification of highly toxic cyanide is also subject to growth inhibition by the cyanide, so the application conditions for purification are limited to low-concentration contamination. For this reason, it has been difficult to detect the activity of microorganisms that purify cyanide compounds by the above detection method.

  In addition, there is a problem that in the total bacteria count, microorganisms that are not subject to monitoring and dead bacteria that do not grow are simultaneously measured. There are plate and MPN methods for measuring the number of viable bacteria. However, since the experimental operation is complicated and requires a test period of about one month, it can be applied to purification projects that require rapid monitoring. There wasn't.

  On the other hand, in order to detect or monitor a bacterial group having a specific function, it is considered desirable to perform comparison and detection at the gene level in terms of accuracy, and in recent years, the 16S rRNA gene Microorganisms are identified using the base sequence. However, since the 16S rRNA gene is a gene possessed by all the microorganisms, it was difficult to detect a specific group of microorganisms by the metabolic mechanism. For this reason, in order to detect a specific microorganism using the 16S rRNA gene as a target, it has been necessary to perform complicated operations such as cloning and sequencing the fragments amplified by PCR one by one.

  As an alternative method, identification, detection and monitoring methods for specific microorganisms targeting genes involved in the purification of pollutants have been developed, and reports have already been made on aromatic dioxygenase genes, vinyl chloride reductase genes, etc. (Patent Documents 3 and 4). However, although a gene for purifying cyanide is already known, there has never been a method for identifying, detecting and monitoring a microorganism for purifying cyanide targeted to the gene.

To date, the following three types cyan purifying mechanism by microorganisms: (i) to consume oxygen completely digested _{(2HCN + 2H 2 O + O} 2 → 2NH 3 + 2CO 2 + 2H 2 O) (ii) Detoxification with an aldehyde (6CN ^- + 6CH ₂ O + 13.5O ₂ → 6CO ₂ + 3N ₂ + 3H ₂ O + 6HCO ₃ ⁻ ); (iii) Detoxification (CN ⁻ + S ₂ O ₃ ^{2 −} → SCN ⁻ + SO ₃ ) by thiosulfate sulfotransferase (hereinafter referred to as “rhodanase”) ^2- ) has been proposed.

  Among these routes, the purification route of (iii) is that in the purification test using artificial groundwater containing cyanide, the addition of thiosulfate to the test culture medium may improve the purification activity by microorganisms. This has been confirmed (Patent Document 2), suggesting that rhodanase is deeply involved in cyanide degradation.

JP 2004-180583 A JP 2006-281053 A JP 9-234070 A JP2007-49946

  An object of the present invention is to provide a method for detecting a microorganism having a cyanide compound resolution quickly, conveniently and specifically.

  The inventors are based on identifying amino acid sequences within the rhodanase gene that are highly conserved among given microorganisms and using this as an indicator for microorganisms with cyanide resolution.

That is, the present invention includes the following features:
(1) A method of detecting a microorganism having a cyanide compound resolution, using a microorganism-derived nucleic acid as a template and the following primers:
(A) Sense primer
5'-GG (T / C / A / G / I) CA (T / C / I) AT (T / C / A / I) CC (T / C / A / G / I) GG (T / C / A / G / I) GC-3 '(SEQ ID NO: 3)
(B) Antisense primer
5 '-(A / G / I) TT (T / C / A / G / I) CCCCA (T / C / I) TC (T / C / A / G / I) (C / G / I) ( T / A / I) CCA-3 '(SEQ ID NO: 4)
(Here, I represents inosine.)
Performing the polymerase chain reaction (PCR) or real-time PCR using the method, and detecting a rhodanase gene amplification product.
(2) The method according to (1), wherein the amplification product is detected by electrophoresis.
(3) The sense primer has the following base sequence:
5'-GGICA (T / C) AT (T / C / A) CCIGGIGC-3 '(SEQ ID NO: 5)
(Here, I represents inosine.)
The method according to (1) or (2), comprising:
(4) The antisense primer has the following base sequence:
5 '-(A / G) TTICCCCA (T / C) TCI (C / G) (T / A) CCA-3' (SEQ ID NO: 6)
(Here, I represents inosine.)
The method according to (1) or (2), comprising:
(5) The method according to any one of (1) to (4), further comprising determining a base sequence of the amplification product.
(6) The method according to (5), further comprising identifying a microorganism based on the determined base sequence.
(7) Using a nucleic acid sequence determined by the method of any one of (1) to (6), the complementary sequence thereof, or a fragment thereof having a length of 15 bases or more, A method for detecting a microorganism having a cyanide compound resolution, comprising detecting a rhodanase gene in the microorganism.
(8) The method according to (7), wherein the detection of the rhodanase gene is carried out using a microarray in which the nucleic acid sequence or a fragment having a length of 15 bases or more thereof is immobilized on a carrier.
(9) The method according to (7), wherein the detection is performed by PCR, real-time PCR, or hybridization.
(10) A primer set for detecting a microorganism having a cyanide compound resolution, which comprises the following primers:
(A) Sense primer
5'-GG (T / C / A / G / I) CA (T / C / I) AT (T / C / A / I) CC (T / C / A / G / I) GG (T / C / A / G / I) GC-3 '(SEQ ID NO: 3)
(B) Antisense primer
5 '-(A / G / I) TT (T / C / A / G / I) CCCCA (T / C / I) TC (T / C / A / G / I) (C / G / I) ( T / A / I) CCA-3 '(SEQ ID NO: 4)
(Here, I represents inosine.)
A primer set as described above.
(11)
The sense primer has the following base sequence:
5'-GGICA (T / C) AT (T / C / A) CCIGGIGC-3 '(SEQ ID NO: 5)
(Here, I represents inosine.)
The primer set according to (10), comprising:
(12) The antisense primer has the following base sequence:
5 '-(A / G) TTICCCCA (T / C) TCI (C / G) (T / A) CCA-3' (SEQ ID NO: 6)
(Here, I represents inosine.)
The primer set according to (10), comprising:

  In the present specification, in the sense and antisense primer sequences, for example, the notation (T / C / A / G / I) is any one base of T, C, A, G and I or two of them. This represents a mix of the above bases.

  By using the detection method of the present invention, microorganisms having cyanide compound resolution can be specifically detected, so that it is possible to determine the suitability of microorganism purification in the bioremediation process and to monitor the microorganism purification activity. become.

Rodanaze is an enzyme publicly known to catalyze the reaction to thiocyanate salt and sulfite from thiosulfate and cyanide. Therefore, the “microorganism having cyanide decomposing ability” used in the present invention refers to a microorganism that produces rhodanase.

  Hereinafter, a method for detecting a microorganism having a cyanide compound resolution according to the present invention and a primer set for use in the method will be described.

  The method for detecting a microorganism having a cyanide-decomposing ability of the present invention is based on identification of an amino acid sequence in a rhodanase gene that is highly conserved among given microbial genera. Therefore, the method for detecting a microorganism having a cyanide compound resolution according to the present invention uses a polymerase chain reaction (hereinafter simply referred to as “PCR”) using a nucleic acid derived from a microorganism as a template and a nucleic acid sequence encoding the conserved amino acid sequence as a primer. Or real-time PCR to detect the rhodanase gene amplification product.

  Here, as used herein, the term “detection” determines not only whether a particular microorganism is present in a sample, but also how much a particular microorganism is present in a sample. (Ie, quantitative detection).

  The inventors have sequences that are highly conserved among the rhodanase genes of different microorganisms, including the genus Nocardioides, Arthrobacter, Mycobacterium, Pseudomonas, (a) amino acid sequence represented by Gly-His-Ile-Pro-Gly-Ala (SEQ ID NO: 1) and (b) amino acid represented by Trp-Thr-Glu-Trp-Gly-Asn (SEQ ID NO: 2) The sequence was found.

  The nucleic acid sequences corresponding to the amino acid sequences are as follows:

  Based on the codons corresponding to the core amino acids constituting the amino acid sequences of SEQ ID NOs: 1 and 2, the following sense primer and antisense primer can be designed, respectively:

  As shown above, the third base of the codon corresponding to C-terminal alanine is deleted in the sense primer. In addition, in the sequence of the 17-mer sense primer and 18-mer antisense primer, N, R, S, Y, W, H in addition to the bases of A, G, C, and T at locations where multiple bases are specified. You may insert the mixed base indicated by (2) or inosine (I) which is a universal base.

  Therefore, in the detection method of the present invention, a rhodanase gene amplification product can be detected by performing PCR using a nucleic acid derived from a microorganism as a template, SEQ ID NO: 3 as a sense primer, and SEQ ID NO: 4 as an antisense primer. .

  Since the sense primer specified by SEQ ID NO: 3 includes a portion that designates a plurality of bases, a plurality of types of nucleic acid sequences can be specified, but in the present invention, only one of them may be used, or a plurality You may use in the form of the random primer containing a nucleic acid sequence of a kind. The same applies to the antisense primer of SEQ ID NO: 4.

  In the detection method of the present invention, a nucleic acid derived from a microorganism as a template can be prepared according to a conventional method, and it is not necessary to use a special method. For example, template DNA can be obtained by obtaining total RNA from a microorganism disruption solution according to a conventional method, collecting mRNA using an oligo dT column, and performing cDNA synthesis in the presence of reverse transcriptase.

  The primers used in the present invention include, for example, cloning of appropriate sequences and cleavage by restriction enzymes, phosphotriester method (see, for example, Narang et al., 1979, Meth. Enzymol., Vol. 68, p90-99), phosphodiester Method (see, for example, Brown et al., 1979, Meth. Enzymol., Vol. 68, p109-151), ethyl phosphoramidite method (see, for example, Beaucage et al., 1981, Tetrahedron Lett., Vol. 22, p1859-1862), etc. It can be directly synthesized by this method. The target primer may be synthesized by using a commercially available automatic DNA synthesizer.

  In the detection method of the present invention, PCR can be performed according to a conventional method. PCR is typically 20 to 40 cycles, with one cycle of denaturation, annealing and extension in the presence of microorganism-derived template DNA, the above-mentioned sense and antisense primers, four bases (dNTP), and a heat-resistant DNA polymerase. To implement. Denaturation is a process for dissociating double-stranded DNA into single strands, and is usually performed at 94 to 96 ° C. for 15 seconds to 5 minutes. Annealing is a process of annealing a primer complementary to a single-stranded template DNA at a temperature below the Tm value set according to the primer used, for example, 50 to 65 ° C., for 30 seconds to 2 minutes. Perform the process. The extension reaction is a reaction for extending a primer along the template DNA, and is usually treated at 72 ° C. for 30 seconds to 10 minutes. The Tm value can be appropriately set by those skilled in the art based on the base sequence of the primer used. In the method of the present invention, in order to guarantee specific binding of the primer, it is preferable to use a temperature of annealing of Tm value −5 ° C. or higher.

  In the detection method of the present invention, the method for detecting the rhodanase gene amplification product is not particularly limited. For example, rhodanase can be detected by electrophoresing the amplified DNA fragment, staining the electrophoresed DNA fragment with, for example, ethidium bromide, and verifying the presence or absence of the fragment of the expected size.

  When PCR is performed using this primer combination, the length of a DNA fragment expected to be amplified is about 700 bp. By using PCR in this way, even when the number of microorganisms having a cyanide compound resolution as a detection target is considerably small, the microorganisms can be detected with high sensitivity and specificity.

  In addition, by performing real-time PCR using this primer combination, it is possible to quantitatively detect microorganisms having cyanide compound resolution. Real-time PCR is a technique for quantifying DNA serving as a template based on amplification rate by measuring amplification by PCR over time. Real-time PCR can be performed according to a conventional method, for example, a method of performing PCR by adding a reagent that fluoresces by binding to double-stranded DNA (Incalator: SYBR (registered trademark) Green I), 5 ′ end Can be carried out by a method of adding an oligonucleotide probe modified with a fluorescent substance (such as FAM) and a 3 'end with a quencher substance (such as TAMRA) to the PCR reaction system.

  For an overview of real-time PCR, see, for example, Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions. Biotechnology 11: 1026-1030 .

  The microorganism that can be detected by the detection method of the present invention is any microorganism that produces a rhodanase enzyme, such as, but not limited to, Nocardioides, Arthrobacter, Mycobacterium, Pseudomonas, Listed are bacteria belonging to the genus Thiobacillus, Azotobacter, Ferrobacillus, Acinetobacter, Ferrobacillus, Escherichia, Shigella, Citrobacter, Klebsiella, Enterobacter, Hafnia, Acidithiobacillus, Worinella It is done.

  The detection method of the present invention may further include the step of determining the base sequence of the rhodanase gene product PCR-amplified as described above. Thereby, for example, by referring to the base sequence registered in the gene database such as GenBank or UniGene, it can be determined whether or not it is the target gene, or the microorganism can be determined. There is no restriction | limiting in particular in the determination method of the base sequence used by this invention, For example, it can implement by well-known methods, such as dideoxy method.

  In general, the base sequence of the rhodanase gene is not only significantly different between microbial genera, but also between species of the same genus or strain, and the degree of difference is the base sequence of 16S rRNA when compared between the same species. Therefore, it is not necessary to determine a long base sequence using a plurality of primers unlike 16S rRNA. Therefore, microorganisms can be identified easily and with high accuracy without performing complicated biochemical tests. In addition, the determined base sequence can be used as a probe as it is, and furthermore, a probe with very high specificity can be created by performing multiple comparisons by similarly determining the base sequences of closely related microbial species. It is. These probes can specifically detect the target microorganism even in the natural flora containing many unknown microorganisms. Moreover, since microorganisms can be easily expressed at the gene level, it is possible to describe new species that have not been identified.

  Therefore, the present invention further comprises detecting a rhodanase gene using the nucleic acid sequence determined by the above-mentioned method, the complementary sequence thereof, or a partial sequence thereof. Provided is a method for detecting a microorganism having compound resolution.

  Here, the “partial sequence” means at least 15 bases, preferably at least 20 bases, more preferably at least 25 bases, such as at least 30 bases, at least 50 bases, of the base sequence encoding rhodanase or its complementary sequence, Refers to a fragment of at least 100 bases in length.

  In this method, when a probe having a relatively high specificity can be designed, direct detection or quantitative detection by various hybridization methods such as Southern hybridization and dot hybridization in addition to PCR and real-time PCR can be performed. Is possible.

At that time, it is preferable to label the nucleic acid sequence with a radioisotope, an enzyme, a fluorescent substance or the like. This makes it possible to easily and easily detect a microorganism having a cyanide compound resolution. Examples of the substance for labeling the nucleic acid sequence of the present invention include, but are not limited to, radioisotopes and fluorescent substances. For example, ³² P, alkaline phosphatase, FITC, rhodamine, fluorescamine, dansyl, biotin, digoxigenin, tetramethyl Examples thereof include rhodamine (TAMRA) and derivatives thereof.

  In the detection method of the present invention, the detection of the rhodanase gene can also be carried out using a DNA microarray in which the sequenced nucleic acid sequence or a part thereof is immobilized on a carrier. Examples of the carrier that can be used here include, but are not limited to, flat substrates such as glass, metal, and various plastic resins. The immobilization method is not limited to this. For example, an adsorption immobilization method using the charge of the substrate and the charge of the nucleic acid, or coating the surface of the substrate with poly-L-lysine or the like to increase the immobilization efficiency. Improving fixation methods are listed. In this case, the nucleic acid derived from the microorganism to be detected can be fluorescently labeled with, for example, sianin.

  The present invention further provides a primer set comprising a combination of a sense primer represented by SEQ ID NO: 3 or 5 and an antisense represented by SEQ ID NO: 4 or 6. This primer set may be in the form of a random primer that includes a plurality of nucleic acid sequences as described above, both for the sense primer and the antisense primer.

  In this example, an Fw primer (5) prepared based on the sequence of the sense primer shown in SEQ ID NO: 3 in the genomic DNA of Nocardioides sp. '-GGICAYATHCCIGGIGC-3': SEQ ID NO: 5) and Rdanase gene using Rv primer (5'-RTTICCCCAYTCISWCCA-3 ': SEQ ID NO: 6) prepared based on the sequence of the antisense primer shown in SEQ ID NO: 4 A PCR amplification test was performed. Similar tests were performed on genomic DNA of Escherichia coli JM109 and Thermotoga maritime, which do not have the ability to purify cyanide. The composition of the PCR reaction solution is as shown below.

  In the PCR reaction, the reaction solution was first subjected to a denaturation reaction at 96 ° C. for 3 minutes. Subsequently, a series of reactions was carried out for 30 cycles, with a denaturation reaction of 96 ° C. for 30 seconds, an annealing reaction of 50 ° C. for 30 seconds, and an extension reaction of 72 ° C. for 1 minute.

  The DNA fragment amplified by the PCR reaction was applied to a 2% agarose gel, and electrophoresis was performed for 30 minutes by applying a voltage of 100 V using a TAE buffer. Subsequently, the agarose gel after electrophoresis was immersed in an ethidium bromide solution, and the DNA fragments in the gel were stained.

  FIG. 1 shows a photograph of a DNA fragment after agarose electrophoresis and ethidium bromide staining. As can be seen from FIG. 1, only in the test using genomic DNA derived from Nocardioides sp as a template, a DNA fragment was confirmed at around 700 bp, indicating that good amplification of DNA of that size occurred. Since the primer used here was expected to have a DNA fragment of 696 bp in size, experimental results consistent with this were obtained. That is, by using the primer according to the present invention, it was shown that the rhodanase gene involved in cyan purification can be specifically and efficiently detected.

FIG. 1 shows that PCR using the nucleic acid sequence of the present invention as a PCR primer resulted in specific amplification of rhodanase from Nocardioides sp. HG-2 strain.

Claims (6)

A method for detecting a microorganism having a cyanide compound resolution, using a microorganism-derived nucleic acid as a template and the following primers:
(A) Sense primer
5'-GG (T / C / A / G / I) CA (T / C / I) AT (T / C / A / I) CC (T / C / A / G / I) GG (T / C / A / G / I) GC-3 '(SEQ ID NO: 3)
(B) Antisense primer
5 '-(A / G / I) TT (T / C / A / G / I) CCCCA (T / C / I) TC (T / C / A / G / I) (C / G / I) ( T / A / I) CCA-3 '(SEQ ID NO: 4)
(Here, I represents inosine.)
Performing the polymerase chain reaction (PCR) or real-time PCR using the method, and detecting a rhodanase gene amplification product. The sense primer has the following base sequence:
5'-GGICA (T / C) AT (T / C / A) CCIGGIGC-3 '(SEQ ID NO: 5)
(Here, I represents inosine.)
The method of claim 1 , comprising: The antisense primer has the following base sequence:
5 '-(A / G) TTICCCCA (T / C) TCI (C / G) (T / A) CCA-3' (SEQ ID NO: 6)
(Here, I represents inosine.)
The method according to claim 1 or 2 , comprising: A primer set for detecting microorganisms having cyanide-decomposing ability, and the following primers:
(A) Sense primer
5'-GG (T / C / A / G / I) CA (T / C / I) AT (T / C / A / I) CC (T / C / A / G / I) GG (T / C / A / G / I) GC-3 '(SEQ ID NO: 3)
(B) Antisense primer
5 '-(A / G / I) TT (T / C / A / G / I) CCCCA (T / C / I) TC (T / C / A / G / I) (C / G / I) ( T / A / I) CCA-3 '(SEQ ID NO: 4)
(Here, I represents inosine.)
A primer set as described above. The sense primer has the following base sequence:
5'-GGICA (T / C) AT (T / C / A) CCIGGIGC-3 '(SEQ ID NO: 5)
(Here, I represents inosine.)
The primer set according to claim 4 , comprising: The antisense primer has the following base sequence:
5 '-(A / G) TTICCCCA (T / C) TCI (C / G) (T / A) CCA-3' (SEQ ID NO: 6)
(Here, I represents inosine.)
The primer set according to claim 4 or 5 , comprising:

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