JP2009079968A - Sample collection method and sample collection device using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample collection method improved in quantitativeness and reproducibility not affected by the collecting technique of a worker, and a sample collection device using the same. <P>SOLUTION: The sample collection device is equipped with a gelled member comprising an agar gel with a gel concentration of 2.5-7.5 w/v%, a holding part for holding the gelled member in a detachable manner and a support part for pressing the gelled member connected to the holding part to a biosample under a pressure of 100-700 g/cm<SP>2</SP>. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sample collection device and a sample collection method using the same, and more specifically, can be suitably used for collection of microorganisms, viruses, and biomolecules.

  Food poisoning, mainly caused by the food industry, is a serious social problem. Many of the causes of food poisoning are caused by the proliferation of microorganisms generated during the processing of meat and food and the contamination of the food with the microorganisms coming into contact with the food.

  In order to prevent food poisoning, it is always necessary to maintain hygiene at the food processing plant. Therefore, in the food industry, it is necessary to check the hygiene level of a food processing plant by measuring the presence of bacteria remaining on cutting boards, tableware, and the like, and food residues serving as a hotbed. Conventionally, the hygiene level has been evaluated by measuring the amount of bacteria by a method called food stamp method or membrane filter method. Common to both the food stamp method and the membrane filter method is that the amount of bacteria is measured by culturing the captured bacteria after capturing the microorganisms. However, with this method, it takes several days until the test result is known, and bacteria cannot be measured in real time.

In addition, food poisoning can be transmitted not only from live bacteria but also from dead bacteria, whereas culture methods can detect only live bacteria. Therefore, focusing on adenosine triphosphate (hereinafter referred to as ATP) common to living organisms, a method of indirectly measuring the amount of bacteria by measuring the amount of ATP has been developed. In this measurement method, since an enzyme reaction is used, the measurement result can be calculated quickly. Therefore, it is an effective technique for quick measurement of food hygiene on the spot. Such ATP measuring devices are sold by many companies, but as a sampling method, sampling with a cotton swab is widely performed. As methods other than cotton swabs, there are a method of collecting a sample using wool or nitrocellulose, which is a material similar to a cotton swab, and a method using a film (see Patent Document 1 and Patent Document 2).
Special table 2003-535318 gazette JP-T-2006-509502

  However, in the above-described conventional configuration, since the sample is collected manually, it is likely to be affected by the operator's sampling skill, and it is easy to cause sampling omissions and variations in the collected amount. Had the problem of decreasing.

  The present invention solves the above-described conventional problems, and an object thereof is to provide a sample collection method having good quantitativeness and reproducibility, and a sample collection apparatus using the same, regardless of an operator's collection skill.

In order to solve the above-mentioned conventional problems, the sample collection method and the sample collection apparatus using the same of the present invention push the gel-like member against the biological sample to be measured with a force of 100 g / cm ² or more and 700 g / cm ² or less. A part of the biological sample is collected by contact.

  Furthermore, a sample collection method and a sample collection apparatus using the same according to the present invention include a gel-like member, a holding portion that removably holds the gel-like member, and the gel-like member connected to the holding portion. And a support for pressing against the biological sample with pressure.

  According to the sampling method of the present invention and the sampling device using the same, the bacteria on the measurement object can be stably stabilized regardless of the skill of the operator by pressing the gel-like member against the measurement object with a constant pressure. Can be collected. Therefore, it is possible to perform measurement with good quantitative and reproducibility.

  In the following, embodiments of the sampling apparatus of the present invention will be described in detail with reference to the drawings.

(Embodiment 1)
FIG. 1 shows the structure of a sampling apparatus according to Embodiment 1 of the present invention. In FIG. 1, a gel holding portion 113 capable of detachably holding the gel-like member 114, a support portion 112 that supports the holding portion 113, and an upper cover surface that is parallel to the upper cover surface a101 and the upper cover surface a101. an upper cover 110 having a b103, supporting the support portion and having a shape enveloping a part of the support portion 112, and having a groove that can be joined to one end of the support portion; and a sampling device main body surface A102 , B104, C105, and a storage unit 109 having a bottom surface of the collection device main body 106, and a support portion 109 through which the support portion 112 can penetrate from the collection device main body surface A102 to the collection device main body surface C105. The collecting device main body 116 having a through hole 108 and a spring 111, and the spring 111 slides on the support through hole 108. Possible in a state in which through the supporting portion 112 penetrating, are mounted within a cavity 107.

  Further, the distance from the upper cover surface a101 to the sampling device main body surface A102 is S1, the distance from the upper cover surface b103 to the sampling device main body surface B104 is S2, and the sampling from the gel-like member sampling plane 115 is performed. When the distance to the apparatus main body bottom surface 106 is S3, the gel-like member collection plane 115 can protrude from the collection apparatus main body bottom surface 106 by adopting a configuration in which the relationship of S1 ≧ S2> S3 is established. On the other hand, it becomes possible to press, and a sample can be collected.

  The gel-like member 114 in the present embodiment may be any material that can collect a biological sample by utilizing the adsorptivity of the gel. Examples of biological samples include microorganisms, viruses, enzymes, and biomolecules. A biomolecule is a nucleotide (polynucleotide (protein) in which nucleic acids and amino acids are linked) that is a nucleotide and a conjugate thereof. Since an agar gel has characteristics that satisfy these uses, in this example, Agar gel was used for the gel-like member 114.

  The rigidity of the gel-like member and the adsorption force for collecting the specimen (ATP) are closely related to the gel concentration. In order to obtain the rigidity and adsorption force of the gel-like member suitable for the purpose of this example, the following gel-like member was produced and tested.

  The agar gel was prepared by using a known method to prepare an agar gel having a concentration of 0.5 w / v% to 7.5 w / v%. There is an upper limit to the agar gel concentration, and a gel exceeding 7.5 w / v% could not be produced. The produced agar gel was cut into a cylindrical shape having a diameter of 30 mm and a height of 15 mm, which is the size of the gel-like member 114 of this example, to produce a cylindrical gel member.

An experiment was conducted using this cylindrical gel member, and a gel concentration showing suitable rigidity was obtained. The cylindrical gel member was pressed against a model sample (details will be described later) coated with an ATP solution, and a uniform pressure was applied to the upper part of the cylindrical gel member for 3 seconds. This operation was repeated 10 times for the same agar gel. The range of applied pressure was 70 to 2000 g / cm ² . The determination of whether or not the rigidity was acceptable was evaluated as “◯” when it was able to withstand 10 repetitions, “Δ” when it was 1 or more and less than 10 times, and “×” when it was not satisfied once and its shape was broken. The results are shown in Table 1.

As is apparent from Table 1, with agar having a gel concentration of 1% or less, the cylindrical gel member collapsed even with a slight pressure, and ATP as a specimen could not be collected. Generally, when the gel concentration is increased, the rigidity of the cylindrical gel member is increased. In this experiment, when the gel concentration was 5 w / v% or more, it was able to withstand a pressing pressure of up to 1250 g / cm ² . When the gel concentration was 2.5 w / v% or more, it was able to withstand a pressing pressure of up to 700 g / cm ² . Since the pressing pressure in the conventional sample collection method using a cotton swab is about 300 g / cm ² , the conventional pressing pressure can be sufficiently satisfied if the gel concentration is 2.5 w / v% or more.

Next, a gel concentration having a suitable adsorption force for the specimen was determined by experiment. The gel concentration of the gel member was 2.5 w / v%, 5 w / v%, 7.5 w / v%. The shape of the agar gel as the gel-like member is the same as that described above, and a model sample obtained by applying 30 μL of an ATP solution prepared to a concentration of 10 ⁻⁵ M on a glass plate was used. Similar to the method for obtaining the rigidity of the gel-like member described above, the gel-like member is pressed against the model sample for 3 seconds with a pressing force of 70 to 700 g / cm ² to adsorb the ATP on the model sample to the gel-like member. . The gel-like member after adsorption was separated from the gel holding part 113 and extracted with 200 μL of pure water. 100 μL of the test solution taken out from the extract and 100 μL of ATP measurement reagent (Lucifer 250 plus, manufactured by Kikkoman Corporation) were mixed to cause the reaction of (Chemical Formula 1).

The amount of light emitted at this time was measured with an ATP collection inspection device (Lumitester PD-10N, manufactured by Kikkoman Corporation). This luminescence amount was defined as the ATP recovery amount. In order to compare the light emission amount under each condition, the light emission amount obtained when the pressing pressure was 700 g / cm ² with a gel-like member having a gel concentration of 7.5% was taken as 100%, and the relative value was calculated. The results are shown in Table 2.

As is clear from Table 2, the ATP recovery amount is more strongly influenced by the pressing pressure than the gel concentration. That is, it can be seen that when the pressing pressure is less than 100 g / cm ² , the amount of ATP recovered is greatly reduced.

From the results of Tables 1 and 2, the preferred pressing force on the gel-like member in this example is 100 to 700 g / cm ² , and the gel concentration is 2.5 w / v% to 7.5 w / v%. is there.

Next, the variation in sample collection amount between the sample collection apparatus of the present invention and the conventional collection method using a cotton swab was compared. The gel concentration of the gel-like member of the sampling device of the present invention was 7.5 w / v%, and the pressing pressure was 300 g / cm ² . As a model sample, 30 μL of an ATP solution having an ATP concentration of 10 ⁻⁹ M was prepared and applied on a glass substrate. Using the sample collection apparatus of the present invention and the conventional method, ATP was collected from the model sample five times, and the amount collected was measured.

  The amount of ATP collected is measured by extracting the collected gel-like member or cotton swab with 200 μL of pure water, and using 100 μL of the test solution taken out from the extract and 100 μL of ATP measurement reagent (Lucifer 250 plus, manufactured by Kikkoman). The reaction of (Chemical Formula 1) was caused by mixing. The amount of light emitted at this time was measured with an ATP collection inspection device (Lumitester PD-10N, manufactured by Kikkoman Corporation). FIG. 2 shows a relative comparison of the light emission amounts at the time of other measurements, with the light emission amount showing the maximum value among the five measurement times as 100%. As shown in FIG. 2, some of the conventional cotton swab collection methods show a reduction of up to 70%, but the collection device of the present invention shows only a reduction of up to 20% and shows little variation. ing. In this case, the sample having a higher concentration had a smaller variation and never dropped below 90%.

(Embodiment 2)
FIG. 3 is a configuration diagram of a sample collection device according to Embodiment 2 of the present invention. In FIG. 3, a contraction spring 204 is installed between the sample collection device main body 116 and the support unit 112 to prevent the gel holding unit 113 from touching the collection surface before collection, and the stopper 203 is It is located between the upper surface 205 of the gel holding part 113 and the lower surface 204 of the support part 112 that supports the gel holding part 113, and is provided to control the operating range of the support part 112. A difference from the configuration is that a pipe 202 is provided to connect the pump 201, the pump 201, and the collection device main body 116 in order to operate the support portion by air pressure.

(Embodiment 3)
FIG. 4 is a configuration diagram of a sample collection device according to Embodiment 3 of the present invention. In FIG. 4, the holding portion 113 that holds the gel-like member 114, the support portion 112 that supports the holding portion 113, the support portion through-hole 108 through which the support portion 112 can pass, and the holding portion 113 can be fitted. And a main body 116 having a stopper through-hole 302 through which a stopper 301 for fixing the accommodating portion 109 and the holding portion 113 can be passed, and a stopper 203 for controlling the working distance of the holding portion. Is different from the configuration of the first embodiment in that the support portion 112 is lifted, and the holding portion 113 is engaged with the sampling device main body 116 by the holding portion stopper 301. The gel-like member 114 is in contact with the sampling surface with the weight of the holding portion 113 itself when the holding portion stopper 301 is opened.

  The sampling device according to the present invention and the sampling method using the sampling device have a capability of eliminating a sampling leakage portion because a sampling area can be fixed by using a gel-like member, and a sampling method capable of collecting a certain amount of sample. It is useful as an apparatus and a sampling method using the apparatus.

The figure which shows the sample-collecting apparatus in Embodiment 1 of this invention Reproducibility evaluation chart of ATP collection amount in the present invention The figure which shows the sample-collecting apparatus in Embodiment 2 of this invention The figure which shows the sampling device in Embodiment 3 of this invention

Explanation of symbols

101 Upper cover surface a
102 Sampling device body surface A
103 Upper cover surface b
104 Sampling device body surface B
105 Sampler surface C
106 bottom surface of sampling device body 107 cavity portion 108 through hole for supporting portion 109 storage portion 110 upper cover 111 spring 112 supporting portion 113 gel holding portion 114 gel-like member 115 gel-like member collecting plane 116 sampling device body S1 from surface a to surface A Distance S2 Distance from surface b to surface B S3 Distance from gel-like member collecting plane to surface D 201 Pump 202 Pipe 203 Stopper 204 Contraction spring 301 Holding part stopper 302 Stopper through hole

Claims (14)

A sample collection method comprising collecting a part of the biological sample by pressing the gel-like member against a biological sample to be measured with a force of 100 g / cm ² or more and 700 g / cm ² or less. The sampling method according to claim 1, wherein the gel-like member has a gel concentration of 2.5 w / v% or more and 7.5 w / v% or less. The sample collection method according to claim 1, wherein the gel-like member has a flat surface pressed against the biological sample. The sampling method according to claim 1, wherein the gel-like member is an agar gel. The sample collection method according to claim 1, wherein the biological sample is adenosine triphosphate. A gel-like member;
A holding part for detachably holding the gel-like member;
A support unit for pressing the gel-like member connected to the holding unit against the biological sample with a predetermined pressure;
A sampling apparatus comprising: The sampling apparatus according to claim 6, wherein the predetermined pressure is in a range of 100 g / cm ^{2 to} 700 g / cm ² . The sample collection device according to claim 6 or 7, wherein the gel-like member has a flat surface pressed against the biological sample. The sampling device according to any one of claims 6 to 8, wherein the gel member has a gel concentration of 2.5 w / v% or more and 7.5 w / v% or less. The sampling device according to claim 6, wherein the gel-like member is an agar gel. 11. The sample collection device according to claim 6, wherein the support unit presses the gel member against the biological sample using an elastic force of an elastic member. The sample collection device according to claim 6, wherein the support unit presses the gel member against the biological sample using air pressure supplied from an external pump. The said support part is equipped with the weight in the said holding | maintenance part in the said support part, The said gel member is pressed against the said biological sample by dropping the said holding part, The Claim 13 characterized by the above-mentioned. Sampling device. The sample collection device according to claim 6, wherein the biological sample is adenosine triphosphate.

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