JP2005265680A - Examination method of legionella bacteria in bathtub water - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an examination method of Legionella bacteria in bathtub water having precision equal to that of a conventional culture method, especially extremely low in false negative ratio, taking no labor even in the extraction of a gene from water to be examined and capable of being performed extremely rapidly. <P>SOLUTION: The examination method of Legionella bacteria in bathtub water has a process (a) for concentrating water to be examined a process (b) for heat-treating the concentrated water to be examined under an alkaline condition, a process (c) for amplifying the gene of Legionella bacteria in the heat-treated sample using an LAMP method and a process (d) for detecting the amplified gene. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for examining Legionella in bath water, and relates to a method for examining Legionella that can obtain an equivalent result in an extremely short time compared to a culture method.

  Legionella is known as the causative microorganism of Legionella disease, and is a Gram-negative bacterium widely inhabiting the natural world and artificial water environment. Inhalation of aerosols such as cooling tower water, hot spring water, and circulating bath water contaminated with Legionella spp. May infect Legionella spp. And cause Legionellosis. Therefore, “new edition legionellosis prevention guideline” (Non-patent Document 1) is established, and it is required to control Legionella spp. In these environmental waters.

  In particular, regarding bath water, Legionella spp. Are not detected according to “Guidelines for water quality standards in public baths” (February 14, 2003, Ministry of Health, Labor and Welfare Health Bureau Director's Notification No. 0212004, Non-Patent Document 2) (10 CFU / 100 mL) Less frequently), and at a certain frequency, inspections are required to see if these standards are met.

  However, the examination of Legionella is carried out by the culture method, for example, as shown in the flowchart (extract) of the plate culture method using the cooling centrifugal concentration method described in “New Legionellosis Prevention Guidelines” in FIG. In addition, the culture takes 5 to 7 days.

However, bath water is water that comes into direct contact with the human body, and when Legionella spp. Are present in the bath water, it is necessary to detect it reliably and at the same time, it is necessary to know the result quickly.
“New Legionellosis Prevention Guidelines” Supervision Ministry of Health and Welfare Life Sanitation Bureau Planning Section, Publisher: (Building Management Education Center, November 1999, first edition issued) “Guidelines for water quality standards in public baths” (February 14, 2003, Ministry of Health, Labor and Welfare, Director General of Health Bureau No.0214004, Non-Patent Document 2)

  The present inventors tried to apply a genetic test method to the test for Legionella spp. Here, since the genetic test method can determine the detection / non-detection of bacteria in about 2 to 10 hours, it is expected that the time can be significantly reduced.

  Nucleic acid amplification and detection methods such as PCR, LAMP, and PALSAR are generally known as genetic testing methods. Among them, nucleic acid amplification methods with high nucleic acid amplification efficiency and the fastest amplification are possible. The target gene can be amplified even if the target gene is very small or there is some reaction inhibitory substance. Therefore, the LAMP method was examined.

  In general, in gene testing methods, it is necessary to extract genes from water to be tested. As such a gene extraction method, it is common to perform a series of operations combining (1) cell lysis, (2) deproteinization and carbohydrate removal, (3) separation and concentration, (4) washing purification, and the like. It is.

  For cell lysis, cell wall lytic enzymes such as lysozyme and achromopeptidase and proteolytic enzymes such as proteinase K are used, or the cells are destroyed using a surfactant such as alkali or SDS. In some cases, physical destruction may occur using beads or ultrasonic waves.

  Extraction by the phenol / chloroform method has been most commonly used for deproteinization and decarburization. In the separation / concentration operation, the nucleic acid is precipitated from an aqueous liquid containing the nucleic acid with isopropyl alcohol, ethanol, or the like and separated and concentrated. In washing and purification, usually, ethanol is used to remove impurities from the separated and concentrated nucleic acid for purification.

  In addition, many kits that combine these methods to enable easy gene extraction are commercially available, but in any case, they are complicated and require time-consuming processing. In the treatment, the more the number of steps, the lower the yield of the target gene to be detected, which increases the possibility of false negatives (determining negative even when bacteria are actually present). Further, if the gene extraction operation is simplified, the yield of the target gene to be detected is increased, but impurities that inhibit the gene amplification reaction cannot be separated, and this impurity is also considered to cause false negatives.

  The present invention improves such problems, that is, a test method for Legionella genus bacteria in bath water, which has the same accuracy as the conventional culture method, in particular, the false negative rate is extremely low, It is an object of the present invention to provide a method for examining Legionella spp. In bath water that can be carried out very quickly and without trouble in gene extraction from water to be examined.

In order to achieve the above object, the present invention is a method for examining Legionella in bath water having the following steps (a) to (d) as described in claim 1.
(A) a step of concentrating water to be inspected;
(B) a step of heat-treating the concentrated water to be examined under alkaline conditions (hereinafter also referred to as “alkaline heat extraction step”),
(C) a step of amplifying Legionella gene in the heat-treated sample using the LAMP method,
(D) A step of detecting the amplified gene

  The method for testing Legionella in the bath water of the present invention is a test method for Legionella in the bath water according to the above-described configuration, and with a comparable accuracy to the conventional culture method, in particular, the false negative rate is extremely low, It is a method for testing Legionella spp. In bath water that can be performed very quickly and without much effort in gene extraction from water to be tested.

  The present invention is a method for examining Legionella spp. In bath water, and the water to be tested is bath water, and water that contains many contaminants such as cooling tower water has a sufficient correlation with the culture method. I can't get it. However, with respect to water such as pool water, it is easy to obtain a correlation with the culture method by this inspection method, and application to such a clean water system is possible.

  In the present invention, the step of concentrating the test target water (step (a)) is specifically a step of concentrating and recovering bacteria in the test target water, and is a normal concentration used for concentration of the test target water. The method can be used. In other words, filtration concentration method, centrifugal concentration method, magnetic bead method, adsorption concentration method using porous material and ferrite, etc. are mentioned, but operation is easy and large equipment such as cooling centrifuge is unnecessary. It is preferable to use a filtration concentration method.

  Here, since the number of viable Legionella bacteria is generally small in the case of bath water, it is preferable to concentrate at least 100 times or more, more preferably 1000 times or more with respect to the water to be examined.

  In the present invention, in the step of heat-treating the water to be examined concentrated in step (a) under alkaline conditions (step (b)), the cells are destroyed and the internal genes are extracted. At this time, other than the method of performing the heat treatment under alkaline conditions, the operation becomes complicated and the yield of the target gene is lowered, so that it is difficult to obtain a test result equivalent to the culture method.

  In step (b), a caustic alkali such as sodium hydroxide or potassium hydroxide is added and heated to pH 11 or higher. The temperature at this time is preferably 90 ° C. or higher. The heat treatment time may be a time sufficient for the cells to break and the gene to elute in the solution, but it is usually preferably 10 minutes or longer. In addition, after completion | finish of (b) process, it adjusts to pH suitable for gene amplification using the LAMP method which is the next process as needed.

  In the step of amplifying the Legionella gene in the sample heat-treated in step (b), which is step (c) in the present invention, it is necessary to use LAMP method. In gene amplification methods other than the LAMP method, it is difficult to keep the false negative rate low, and the coincidence rate with the results of the culture method is extremely low, so that a test result having sufficient reliability cannot be obtained.

  In the present invention, LAMP (Loop-mediated thermal amplification) method refers to Tokushu WO00-28082, Tokushui WO01-034838, JP2001-242169, JP2002-272475, and JP2002-345499. This is a method proposed or described in publications, etc., and has at least two types of inner primers having a sequence complementary to the sequence on the extended strand on the 5 ′ side, and at least two types of outer primers necessary for starting material synthesis And a gene amplification method of amplifying a target gene using a strand displacement type DNA synthase. In the LAMP method, the amplification process is performed at a constant temperature (around 65 ° C.). Therefore, unlike the PCR method involving repeated temperature rise and fall, extremely rapid gene amplification is considered possible.

  In the step (d) of the present invention, that is, in the step of detecting the gene amplified in the step (c), the amplification product is detected by a general agarose gel electrophoresis detection method or LAMP. Specific detection methods include detection of increased turbidity due to poorly soluble pyrophosphate, a byproduct of gene amplification reactions, and hybridization to single-stranded loops of gene amplification products obtained by LAMP reactions For example, a method of measuring the degree of fluorescence polarization of the reaction solution by adding a fluorescently labeled primer can be employed.

  By combining the alkaline heat extraction method and the LAMP method, the present invention has almost no false negatives, has a high coincidence rate with the culture method, and has excellent results in a short period of time. Is the method.

  Examples of the present invention will be described below. The sample used the actual bathtub water collected from 50 places, such as a public bath and a hot spring, as water to be examined.

(Example)
<(A) Process: Concentration of water to be inspected>
After each 500 mL of sample water was filtered using a membrane filter (pore size: 0.45 μm), 50 mL of sterile deionized water was filtered to rinse the membrane filter.

  Place the membrane filter with the filtered trapped substance in a sterilized 50 mL centrifuge tube with screw cap, add 5 ml of sterilized deionized water, and stir for 5 minutes with a vortex mixer to resuspend the captured substance. It was.

<(B) Process: The process heat-processed on alkaline conditions (alkali heat extraction process)>
First, 2 mL of the resuspension obtained above was transferred to a 2 mL microtube and centrifuged at 14,000 × g for 10 minutes at 4 ° C.

  1960 μL of the supernatant was removed, 50 μL of 50 mM sodium hydroxide aqueous solution was added, the microtube was immersed in boiling water (100 ° C.), and heated for 15 minutes to extract the gene outside the cells. Next, the microtube was ice-cooled, neutralized by adding 8 μL of 1M-Tris-HCl buffer (pH 7.0), and centrifuged at 14,000 × g for 10 minutes at a temperature of 4 ° C. The supernatant was used as a sample for gene amplification.

<Step (c): Step of amplifying Legionella gene using LAMP method>
5 μL of the gene amplification sample prepared above was added to 20 μL of the reaction mixture whose composition is shown in Table 1, and reacted at 65 ° C. for 60 minutes.

  The primers used at this time were 8 types of primers shown in Table 1 including 6 regions targeting the 16S rRNA gene of Legionella spp.

<(D) Process: The process which detects the amplified gene>
After 60 minutes from the start of the amplification reaction by the LAMP method, a sample in which the reaction solution became turbid due to magnesium pyrophosphate which is a by-product of the LAMP reaction was determined to be positive for Legionella.

(Comparative Example 1: Examination by LAMP method using gene extraction kit)
In the same manner as in the above examples, however, gene extraction using a commercially available gene extraction kit was performed instead of alkaline heat extraction. That is, for 100 μL of the re-suspension after step (a), gene extraction was performed according to the high-sensitivity protocol using the gene extraction kit EXTRAGEN made by Tosoh Corporation (the operation at this time was 13 steps). Finally, the sample was dissolved in 25 μL of TE buffer to prepare a sample for gene amplification, and then the steps (c) and (d) of the above example were performed.

(Comparative Example 2: Examination by PCR method)
Similar to the LAMP method, the PCR method (polymerase chain reaction method), which is another method belonging to the gene amplification test method, was examined. That is, after performing the steps (a) and (b) in the same manner as in the above example, the PCR method was performed as follows.

  Primers used for PCR were Yamamoto et al. (“Comparison of culture method, fluorescent antibody staining method, PCR method as a method for detecting Legionella spp. In environmental water” (Yamamoto, H., Y. Hashimoto and T. Ezaki (1993) Comparison of detection methods for Legionella species in environmental water by colony isolation, fluorescent antibody staining, and polymerase chain reaction. Microbiol. Immunol., 37, 617-622), targeting 16S rRNA of Legionella spp. For LEG448-A (5′-GAG GGT TGA TAG GTT AAG AGC-3 ′) (SEQ ID NO: 9) and LEG854-B (5′-CGG TCA ACT TAT CGC GTT TGC T-3 ′) (SEQ ID NO: 10) It was.

  The PCR reaction solution was prepared using TaKaRa Ex Taq Hot Start Version (manufactured by Takara Bio Inc.). The composition of the reaction solution was 2.5 μL of 10 × Ex Taq buffer (buffer), 2 μL of d-NTP mix, 0.5 μL of LEG448-A primer solution (10 μM), 0.5 μL in 14.375 μL of sterile distilled water. LEG854-B primer solution (10 μM) and 0.125 μL of Ex Taq polymerase were added, and finally 5 μL of nucleic acid extract was added to make a total volume of 25 μL.

  The thermal cycler was set for 5 cycles, with the initial heat denaturation at 94 ° C. for 90 seconds, followed by 94 ° C. for 30 seconds, 65 ° C. for 60 seconds, and 72 ° C. for 60 seconds. Thereafter, 35 cycles were carried out, with one cycle consisting of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds and 72 ° C. for 60 seconds, and finally a reaction was carried out at 72 ° C. for 4 minutes.

  The amplification product was detected by agarose gel (1.5% by weight) electrophoresis. The gel after electrophoresis is stained with SYBR Green I (BMP), and the presence or absence of a 430 bp amplification product derived from Legionella is observed on a 254 nm transilluminator. I decided.

(Comparative Example 3: Examination by culture method)
The culture method was examined using 1 mL of the resuspension after performing the step (a) in the same manner as in the above Example.

  The culture method was performed using the GVPCα medium according to the flowchart shown in FIG. 2, which was determined according to the flowchart of the plate culture method using the cooling centrifugal concentration method described in the “New Edition Legionellosis Prevention Guidelines” (FIG. 1). . The culture days at this time were 6 days.

  Table 2 shows the results of testing the actual bath water collected from 50 places such as public baths and hot springs, respectively, by the method of the example and the method of the comparative example, as the test target water. In Table 2, “Concordance rate with culture method” indicates the rate at which the results for each test water match the results of the culture method and the results of each method, and “false negative rate” is the result of the culture method. The test water whose result was “positive” was the ratio that the result by each method was determined to be “negative”, and the “false positive rate” was the water to be tested whose result by culture method was “negative” The ratio at which the result of each method was determined to be “positive” is shown respectively.

  Moreover, about the cooling water of the cooling tower collected from 50 places, the result investigated by the method similar to an Example and the comparative example 3 is described together in Table 2 as reference data.

  According to Table 2, the method for examining Legionella spp. According to the present invention shows that the combination of the alkaline heat extraction step and gene amplification by the LAMP method has the highest coincidence rate with the culture method in the bath water test, and in fact, Legionella The false negative rate at which false negatives are judged as “safe” for Legionella spp. Despite the presence of the genus is 0%.

  On the other hand, in the combination of gene extraction with a commercially available kit and LAMP method, the agreement rate with the culture method is low, and the probability of false negative is 18 despite the fact that gene extraction takes a very complicated time. % Is very high, and it can be seen that it is completely unreliable.

  Further, the combination of the alkaline heat extraction step and the PCR method has a false negative rate of 4%, and again, as a test method for Legionella spp., The false negative rate that should be avoided is the highest and lacks reliability.

  On the other hand, in the inspection for the cooling water of the cooling tower in which many contaminants coexist, even the inspection method having a combination of the alkaline heat extraction process and gene amplification by the LAMP method is consistent with the culture method The rate is less than half, and it is judged that this inspection method is not suitable for cooling tower cooling water.

  The present invention is a method for testing Legionella spp. In bath water, which has the same accuracy as the conventional culture method, particularly has a very low false negative rate, and is troublesome in gene extraction from water to be tested. It is an inspection method for Legionella spp. In bath water that can be carried out very quickly without being hung, and can be suitably used as a test method for Legionella spp. For safety management of bath water in public baths, etc. it can.

It is a flowchart (extract) of the plate culture method using the cooling centrifugal concentration method described in "New edition Legionellosis prevention guideline". 10 is a flowchart of an inspection method performed in the examination by the culture method of Comparative Example 3.

Claims (1)

The inspection method of Legionella genus bacteria in bath water characterized by having the process of following (a)-(d).
(A) a step of concentrating water to be inspected;
(B) a step of heat-treating the concentrated water to be examined under alkaline conditions;
(C) a step of amplifying Legionella gene in the heat-treated sample using the LAMP method,
(D) A step of detecting the amplified gene

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