JP2017020807A - Organism-derived stain detection/quantitation method and detection/quantitation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a device for simply and efficiently performing extraction and detection/quantitation of an organism-derived stain without dismantling an inspection object and without giving the inspection object a physical stress that may cause malfunction in the inspection object.SOLUTION: An organism-derived stain detection/quantitation method according to the present invention is for performing the detection/quantitation of the organism-derived stain adhered to the inspection object. The method includes an extraction process of extracting the organism-derived stain by bringing the inspection object into fluid contact with an extract containing a surfactant, and a detection/quantitation process of subjecting the extracted organism-derived stain to detection/quantitation. An organism-derived stain detection/quantitation device 1 according to the present invention comprises extraction means 10 that extracts the organism-derived stain by bringing the inspection object 11 into fluid contact with the extract containing the surfactant, and detection/quantitation means 20 that subjects the extracted organism-derived stain to detection/quantitation. The extraction means 10 includes a container 12 that stores the inspection object 11 and supply means 13 that continuously supplies the extract into the container 12.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a detection and quantification method and a detection and quantification apparatus for biologically derived soil.

In recent years, the problem of nosocomial infection has increased, and reducing the risk of infection has become an essential issue in medical institutions.
Since medical instruments are generally expensive, cleaning and recycling are performed as much as possible. In order to reduce the risk of infection, medical devices with biological dirt such as blood, body fluids, tissue fragments, etc. that may contain bacteria or viruses as infectious agents are usually washed (first step) Cleaning and recycling are performed through two steps of disinfection (second step).

Methods for cleaning (first step) include immersion cleaning, manual cleaning, and mechanical cleaning using an ultrasonic cleaner, a washer-disinfector, and the like.
However, there may be cases where the biologically-derived soil remains on the medical instrument even after the above cleaning. In that case, the effect of disinfection (second step) is not sufficiently exhibited, and the possibility of infection increases. For this reason, it is necessary to check the cleanliness of the medical device after washing.

In recent years, endoscopes have been used from the viewpoints of suppressing complications such as pain, fever, bleeding, and infectious diseases in patients and reducing the economic burden associated with early recovery of patients, early rehabilitation, shortening hospitalization, etc. Many invasive treatments and examinations have been carried out by using laparoscopes, surgery support robots, and the like.
An arm-like instrument used for an endoscope, a laparoscope, a surgery support robot and corresponding to a doctor's hand (for example, EndoWrist (registered trademark), da Vinci forceps, operation forceps used in da Vinci Surgical System) It has a complex internal structure composed of many precision parts to allow complex operations. Moreover, since they are often difficult to disassemble, they are more difficult to clean than ordinary tools and are less likely to remove dirt. In addition, it is difficult to check the degree of dirt removal.
Therefore, there is a method for extracting biologically-derived dirt from the latest medical instruments, particularly for the purpose of evaluating the cleanliness of the latest medical instruments such as endoscopes, laparoscopes, EndoWrist (registered trademark), da Vinci forceps, and operating forceps. It has been demanded.

  As an extraction method for evaluating the cleanliness of operation forceps after cleaning, for example, Non-Patent Document 1 discloses that EndoWrist (registered trademark) collected from each facility is used 10 times after being disassembled into a tip portion and a shaft portion. A method is disclosed in which a disassembled tip portion and an extract (0.2 NaOH / 1% SDS) are put in a test tube, and the residual protein is extracted by repeating immersion at 70 ° C. for 60 minutes and ultrasonic treatment.

  In Non-Patent Document 2, contaminated blood added with Bacillus subtilis ATCC6633 (spore form) is injected twice into the lumen of the da Vinci forceps with a 50 mL syringe, and after a natural drying for 4 hours, a washing process for 45 minutes is performed. Thereafter, a method of extracting the remaining viable bacteria for 5 minutes using a tabletop ultrasonic cleaner is disclosed. In the method described in Non-Patent Document 2, with the tip of the forceps embedded in 100 mL of sterilized water, the remaining viable bacteria are extracted by circulating and injecting the forceps tip twice with a 50 mL syringe and flowing the tip of the forceps. Yes.

  In Non-Patent Document 3, the outer surface of the operation forceps used in the robot-assisted surgery and the protein mass on the forceps surface were measured by wiping with a cocoase containing 0.5 mL of distilled water. About the amount, the operation forceps that have been wiped are inserted into a plastic bag, 100 mL each of distilled water is injected from two flush ports, and the entire operation forceps is filled with warm water while excluding air in the plastic bag. A method is disclosed in which the protein is extracted by putting the solution in a washing tank through a bag, outputting an ultrasonic oscillator, and sufficiently stirring the solution in the plastic bag after 30 minutes.

Satoshi Fujita et al., “Examination of Cleaning Evaluation Method for Surgery Assistive Robot Instrument”, Medical Device Science, Japanese Society of Medical Device, Vol. 84, No. 2, p147 (2014) Furuhata Sadahiko et al., “Verification of cleaning capability with surgical support robot's forceps cleaning rack”, Medical Device Science, Japanese Society of Medical Device, Vol. 84, No. 2, p146 (2014) Yuhei Saito et al., “Stained areas of operation forceps for surgical support robots”, Medical Device Science, Japan Society for Medical Device, Vol. 84, No. 2, p150 (2014)

  However, according to the method described in Non-Patent Document 1, when extracting the residual protein from EndoWrist (registered trademark), it is decomposed into a tip portion and a shaft portion. Therefore, it is difficult to reuse EndoWrist (registered trademark), and the device is reused. From the viewpoint of use, it is not suitable as an extraction method. In addition, since the protein is extracted only from the tip of EndoWrist (registered trademark) and the protein is not extracted from the shaft or the main body, it is difficult to accurately evaluate the cleanliness. In addition, because high-temperature, high-concentration alkaline aqueous solution is used as the extraction liquid and protein is extracted by sonication for a long time, there is a risk of corrosion or failure of the EndoWrist (registered trademark) component having a very fine structure. There is.

  As in the method described in Non-Patent Document 2, sterilized water having low osmotic action and dispersive action is used, and sonication for 5 minutes alone cannot sufficiently extract dirt inside the instrument. In addition, since the protein is extracted by ultrasonic irradiation that is subject to physical stress, the DaVinci forceps may break down.

  In the method of Non-Patent Document 3, since the operation forceps are divided into the outer surface, the forceps surface, and the lumen portion to extract the protein, the work is complicated. Further, since distilled water having a low osmotic action and dispersive action is used for extracting the lumen portion of the operation forceps, dirt inside the instrument cannot be sufficiently extracted. Furthermore, since protein is extracted by ultrasonic irradiation that is subject to physical stress, the operation forceps may be damaged.

  The present invention has been made in view of the above circumstances, and can easily and efficiently extract biologically-derived dirt without disassembling the inspection object and without applying physical stress that causes failure to the inspection object. It is an object of the present invention to provide a method and an apparatus for detecting and quantifying a target.

The present invention has the following aspects.
[1] A method for detecting and quantifying biologically-derived soil adhering to a test object, wherein an extraction liquid containing a surfactant is fluidly contacted with the test object to extract biologically-derived soil; And a detection and quantification step of detecting and quantifying the extracted biologically-derived soil.
[2] The method for detecting and quantifying biologically-derived soil according to [1], wherein the extract is circulated and repeatedly brought into fluid contact with the test object.
[3] The method for detecting and quantifying biologically-derived soil according to [1] or [2], wherein the test object is an instrument that has been cleaned after use.
[4] The method for detecting and quantifying biologically-derived soil according to any one of [1] to [3], wherein the extract has a viscosity of 0.1 to 500 mPa · s.
[5] The inspection object is a manipulator having a liquid inlet, an adapter is attached to the liquid inlet, the extract is injected, and the extract is fluidly contacted with the manipulator [1] to [4] The method for detecting and quantifying biologically-derived soil according to any one of the above.

[6] An apparatus for detecting and quantifying biologically-derived soil adhering to a test object, the extraction means for extracting biologically-derived soil by fluidly contacting the test object with an extract containing a surfactant. And a detection and quantification means for detecting and quantifying the extracted biologically-derived soil, wherein the extraction means includes a container for storing the test object and a supply means for continuously supplying the extract to the container. Detecting and quantifying device for biologically derived dirt.
[7] The extraction liquid discharged from the container is returned to the supply means, and the extraction liquid circulates between the container and the supply means and repeatedly and fluidly contacts the inspection object. Detection and quantification device for soil derived from living organisms.

  According to the detection and quantification method and the detection and quantification apparatus for biologically-derived soil of the present invention, it is simple and efficient to derive from a biological body without decomposing the inspection object and without applying physical stress that causes a failure to the inspection object. Detect and quantify by extracting dirt.

It is a schematic block diagram which shows an example of the detection and quantification apparatus of the biological origin dirt of this invention. It is a perspective view which shows an example of a container. It is a figure which shows the other example of a container, (a) is a top view, (b) is a side view. It is a figure which shows the other example of a container, (a) is a top view, (b) is a side view. (A) is a perspective view which shows an example of an adapter, (b) is sectional drawing which shows the state which mounted | wore the liquid injection port of the manipulator with the adapter shown to Fig.5 (a). (A) is a perspective view which shows the other example of an adapter, (b) is sectional drawing which shows the state which mounted | wore the liquid injection port of the manipulator with the adapter shown to Fig.6 (a). (A) is a perspective view which shows the other example of an adapter, (b) is sectional drawing which shows the state which mounted | wore the liquid injection port of the manipulator with the adapter shown to Fig.7 (a). (A) is a perspective view which shows the other example of an adapter, (b) is sectional drawing which shows the state which mounted | wore the liquid injection port of the manipulator with the adapter shown to Fig.8 (a). It is a perspective view which shows the other example of an extraction means. 2 is a graph showing the relationship between extraction time and protein extraction amount in Example 1.

Hereinafter, the present invention will be described in detail.
"Detection and quantification method of biological soil"
The biologically-derived soil detection and quantification method of the present invention is a method for detecting and quantifying biologically-derived soil adhered to a test object, and includes the following extraction step and detection and quantification step.
In the present invention, “biologically-derived soil” refers to soil adhered by surgery or the like, and examples thereof include blood, body fluid, and tissue pieces.

<Inspection object>
The inspection object is not particularly limited as long as there is a possibility that biologically derived dirt is attached.
Examples of the inspection target include instruments such as medical instruments used for operations, examinations, treatments, and the like, and food instruments used for food production and processing. Moreover, it is good also considering what cleaned after using these instruments, and what performed cleaning and disinfection as a test object. Among these, from the viewpoint of confirming whether or not a tool that has been cleaned after use can be reused, or confirming the appropriateness of the cleaning method for the tool, the object to be inspected may be cleaned after use, or washed and disinfected. The instrument which performed is suitable.

  Examples of the medical instrument include an arm-shaped instrument (hereinafter referred to as a “manipulator”) that is used for an endoscope, a laparoscope, a surgery support robot, and corresponds to a doctor's hand, an insulator, a forceps, a scissors, a suction tube, and a catheter. And injection needles. Among these, the present invention is a medical instrument in which extraction and cleanliness confirmation are difficult, specifically, a medical instrument composed of precise parts and difficult to disassemble (for example, endoscope, laparoscope, manipulator) Among them, it is particularly suitable for a manipulator.

Examples of the manipulator include EndoWrist (registered trademark), da Vinci forceps, operation forceps and the like used in the da Vinci Surgical System.
The manipulator is generally composed of a tip portion, a shaft portion, and a main body portion.
The tip of the manipulator is composed of a jig such as a lever, forceps, and scissors that perform precise operations such as surgery.
The shaft portion of the manipulator connects the main body portion and the tip portion, and has a tubular structure for inserting a wire for driving a jig at the tip portion.
The main body (also referred to as “housing”) of the manipulator has an internal structure made up of precision parts such as wires and pulleys in order to enable operation of the tip. Further, the main body has a liquid inlet (also referred to as “main flash port”) for injecting a cleaning liquid or the like, and after the cleaning liquid or the like injected from the liquid inlet has been transmitted through the main body, It is structured to be discharged from the gap at the tip via With such a structure, the inside of the manipulator can be cleaned after the manipulator is used.

<Extraction process>
The extraction step is a step of extracting biologically derived dirt by bringing a liquid extract containing a surfactant into fluid contact with the test object. Hereinafter, the extract before being brought into contact with the test object is also referred to as “pre-contact extract”, and the extract from which biological soil is extracted is also referred to as “biological soil extract”.

(Extract)
The extract used in the extraction step contains a surfactant.
The surfactant is not particularly limited as long as it has a strong detergency with respect to biologically derived dirt and does not easily corrode the test object. An anionic surfactant, a cationic surfactant, Nonionic surfactants and amphoteric surfactants can be mentioned. These can use 1 type (s) or 2 or more types. Among these, anionic surfactants, nonionic surfactants, and amphoteric surfactants are preferable from the viewpoint of high extraction efficiency of biologically derived soils (particularly proteins) and resistance to corrosion of the test object, and anionic surfactants, nonions A surfactant is more preferred.

  The anionic surfactant is not particularly limited, and examples thereof include soaps having 10 to 22 carbon atoms, alkylbenzene sulfonates having 14 to 24 carbon atoms, higher alcohol sulfates having 10 to 22 carbon atoms, and alkyl groups having 10 carbon atoms. A polyoxyethylene alkyl ether sulfate having 1 to 10 repeating oxyethylene units of a polyoxyethylene group, an α-sulfo fatty acid ester having 10 to 22 carbon atoms, and an α-olefin sulfone having 8 to 18 carbon atoms Examples thereof include acid salts, alkane sulfonates having 10 to 22 carbon atoms, and monoalkyl phosphate esters having 10 to 22 carbon atoms. Among these, from the viewpoint of high extraction efficiency of biologically derived soils (particularly proteins) and being difficult to corrode the test object, soap having 10 to 22 carbon atoms, alkylbenzene sulfonate having 14 to 24 carbon atoms, and 10 to 10 carbon atoms. 22 higher alcohol sulfate ester salt, polyoxyethylene alkyl ether sulfate having 10 to 22 carbon atoms in the alkyl group and 1-10 repeats of polyoxyethylene group oxyethylene units, and α- Preferred are olefin sulfonates, alkane sulfonates having 10 to 22 carbon atoms, higher alcohol sulfates having 10 to 22 carbon atoms, α-olefin sulfonates having 8 to 18 carbon atoms, and alkanes having 10 to 22 carbon atoms. Sulfonates are more preferred, and higher alcohol sulfates having 10 to 22 carbon atoms are particularly preferred. These can use 1 type (s) or 2 or more types.

  The amphoteric surfactant is not particularly limited, and examples thereof include alkylamino fatty acid salts, alkylbetaines, and alkylamine oxides. These can use 1 type (s) or 2 or more types.

  The nonionic surfactant is not particularly limited. For example, higher alcohol alkylene oxide adduct, alkylphenol alkylene oxide adduct, fatty acid alkylene oxide adduct, polyhydric alcohol fatty acid ester alkylene oxide adduct, higher alkylamine alkylene oxide adduct, Fatty acid amide alkylene oxide adduct, polyalkylene glycol type of polyoxypropylene alkylene oxide adduct; glycerol fatty acid ester, pentaerythritol fatty acid ester, sorbitol fatty acid ester, polyhydric alcohol type of sucrose fatty acid ester, and the like. The higher alcohols mentioned here are usually linear or branched unsaturated or saturated higher alcohols having 8 to 22 carbon atoms. The alkylphenol is usually a linear or branched unsaturated or saturated alkylphenol having 6 to 22 carbon atoms. The fatty acid is usually an unsaturated or saturated fatty acid having 10 to 22 carbon atoms. The polyhydric alcohol is usually a polyhydric alcohol having 3 to 12 carbon atoms. The higher alkylamine is usually a linear or branched unsaturated or saturated higher alkylamine having 8 to 22 carbon atoms. Specific examples of the alkylene oxide include ethylene oxide, propylene oxide, 1,2-, 2,3-, 1,3- and 1,4-butylene oxide, and ethylene oxide and propylene oxide are preferable. Further, the alkylene oxides may be the same or different, and if they are different, block addition, random addition, or alternate addition may be used. 1-80 are preferable, as for the addition mole number of alkylene oxide, 2-60 are more preferable, and 5-40 are especially preferable. Among these, higher alcohol alkylene oxide adducts, alkylphenol alkylene oxide adducts, fatty acid alkylene oxide adducts, polyhydric alcohol fatty acids from the viewpoint of high extraction efficiency of biologically derived dirt (particularly proteins) and resistance to corrosion of the test object. Ester alkylene oxide adduct, higher alkylamine alkylene oxide adduct, fatty acid amide alkylene oxide adduct, polyalkylene glycol type of polyoxypropylene alkylene oxide adduct; glycerol fatty acid ester is preferred, higher alcohol alkylene oxide adduct, alkylphenol alkylene Oxide adduct, fatty acid alkylene oxide adduct, polyhydric alcohol fatty acid ester alkylene oxide adduct, higher alkyl amine Emissions alkylene oxide adducts, fatty acid amide alkylene oxide adducts are more preferred. These can use 1 type (s) or 2 or more types.

  The concentration of the surfactant is preferably 0.01 to 10% by mass, more preferably 0.1 to 5% by mass in 100% by mass of the extract from the viewpoint of increasing the extraction efficiency of biologically derived dirt and being economical. .

  As a solvent for diluting the surfactant, water is preferable, and specific examples include tap water, ion exchange water, distilled water, and RO water. These can use 1 type (s) or 2 or more types.

The extract may contain an alkaline agent as long as the test object is not damaged.
Examples of the alkaline agent include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, sodium phosphate, potassium phosphate and the like. These can use 1 type (s) or 2 or more types.
The concentration of the alkaline agent is preferably 10% by mass or less, more preferably 5% by mass or less, and more preferably 0% by mass in 100% by mass of the extract from the viewpoint of enhancing the extraction efficiency of biologically derived dirt and making it difficult to corrode the test object. (Not containing an alkali agent) is particularly preferable.

Moreover, the extract may contain an organic solvent as long as the test object is not damaged.
Examples of the organic solvent include alcohols such as methanol, ethanol and isopropyl alcohol; ethers such as dimethyl ether, ethyl methyl ether and diethyl ether; esters such as methyl acetate and methyl salicylate; ketones such as acetone, methyl ethyl ketone and diethyl ketone Glycols such as ethylene glycol and propylene glycol; glycol ethers such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether and ethylene glycol monobutyl ether; alicyclic hydrocarbons such as cyclohexane and methylcyclohexane; normal hexane, normal heptane Aliphatic hydrocarbons such as benzene, toluene, xylene and the like. These can use 1 type (s) or 2 or more types.

The viscosity of the extract is preferably from 0.1 to 500 mPa · s, more preferably from 0.2 to 100 mPa · s. When the viscosity of the extract is 0.1 mPa · s or more, the permeability of the extract with respect to biologically derived dirt increases. On the other hand, if the viscosity of the extract is 500 mPa · s or less, the extract can easily reach the details of the inspection object. Moreover, when the extraction means to be described later is used to circulate the extract and repeatedly contact the test object, the extract flows smoothly.
The viscosity of the extract can be determined by measuring the viscosity of the extract before contact at a liquid temperature of 20 ° C. using a capillary viscometer such as a Canon-Fenske viscometer.

(Contact method)
In the extraction process, the extract is fluidly contacted with the inspection object. The contact method is not particularly limited as long as the extract is flowing. For example, the test object is accommodated in a container to be described later, and the extract is fluidized in the test object by continuously flowing the extract into the container. The method of making it contact is mentioned.
By bringing the extract into fluid contact with the test object, it is simple and efficient without decomposing the test object and without subjecting the test object to physical stresses such as sonication that cause failure. Biologically derived dirt can be extracted.

  From the viewpoint of efficiently extracting the biologically-derived soil in a small amount, it is preferable to circulate the extract and repeatedly and fluidly contact the test object. Circulating the extract is to repeat the operation of fluidly contacting the biologically derived soil extract that has passed through the test object again with the test object. For example, when the test object is accommodated in the container and the extract is brought into fluid contact, the operation of supplying the biologically derived soil extract discharged from the container to the container again may be repeated.

  When the object to be inspected is a manipulator, it is preferable to attach an adapter to the liquid inlet of the manipulator to inject the extract, and to bring the extract into fluid contact with the manipulator. The extraction liquid injected from the liquid injection port travels through the main body of the manipulator and then is discharged from the gap at the tip through the shaft. Thereby, the living body-derived dirt inside the manipulator can be sufficiently extracted.

When the test object is accommodated in the container and the extract is brought into contact with the test object, the extract is preferably brought into contact with the test object in a state where the container is inclined. By tilting the container, the amount of the extract that stays in the container can be reduced even when the amount of the extract is small.
Moreover, when the material of the container is soft and the form changes due to compression or the like, it is preferable to bring the extract into contact with the inspection object in a state where the inside of the container is decompressed. By reducing the pressure inside the container, the gap between the container and the inspection object is reduced, and the extract can be brought into intensive contact with the inside and the periphery of the inspection object.

  The flow rate of the extract is not particularly limited, but is preferably 10 to 10,000 mL / min, more preferably 20 to 5,000 mL / min in consideration of the extraction efficiency and the load on the test object.

  The amount of the extract used (the amount of the extract) may be adjusted according to the size, surface area, usage, etc. of the test object to be detected, and is not particularly limited, but is obtained by the extraction process. Taking into account the detection of the extracted components in the biologically derived soil extract, it is preferable to select as small an amount as possible.

The temperature of the extract is not particularly limited, but is preferably 0 to 150 ° C., more preferably 10 to 80 ° C., and particularly preferably 30 to 60 ° C. in consideration of the extraction efficiency and the load on the test object.
It is preferable to remove bubbles from the extract in advance by ultrasonic treatment or the like. By removing the bubbles, the effect of extracting the biologically derived dirt is further enhanced.

  The extraction time may be adjusted according to the flow rate, temperature, etc. of the extract, and is not particularly limited. However, from the viewpoint of increasing the extraction efficiency of biologically derived dirt and being economical, it is 0.1 to 100 hours. Is preferable, and 0.5 to 50 hours is more preferable.

<Detection quantitative process>
The detection and quantification step is a step for detecting and quantifying the biologically-derived soil extracted in the extraction step.
The components extracted from the inspection object by the extraction process are dirt adhered by surgery or the like, and are mainly living body-derived dirt. Examples of methods for detecting biologically-derived soil include methods for detecting proteins, nucleic acids, sugars, fats, ATP, hemoglobin, and the like in biologically-derived soils, but there are no particular limitations as long as these are detected. For example, protein detection methods include CBB method, BCA method and the like. Examples of the nucleic acid detection method include a UV method. Examples of the sugar detection method include a phenol sulfuric acid method, a somogi method, a belt run method, an enzymatic method, a method using HPLC, GC, GC-MS, and the like. Examples of the fat detection method include an enzymatic method, a method using HPLC, GC, and GC-MS. A biological luminescence method etc. are mentioned as an ATP detection method. Examples of the hemoglobin detection method include an SLS-Hb method, a cyanmethoglobin method, an orthotolidine method, a guaiac method, and an immunization method. Among these, from the viewpoint of simplicity and accuracy of quantification, the extract is reacted with a protein detection solution containing bicinchoninic acid and copper (II) in an alkaline environment, and the resulting reaction solution is measured at 562 nm with a spectrophotometer. The BCA method for measuring the absorbance of the liquid, the biological luminescence method for contacting the ATP detection solution containing luciferase and the like with the extract, and measuring the luminescence amount of the obtained contact solution, and the extract containing sodium lauryl sulfate (SLS) The SLS-Hb method is preferred in which a hemoglobin coloring reagent is reacted and the resulting reaction solution is measured for absorbance at 540 nm with a spectrophotometer.

<Effect>
Since the biologically-derived soil detection and quantification method of the present invention described above extracts the biologically-derived soil by bringing the extract into fluid contact with the inspection object, the inspection object is not decomposed and the cause of the failure. Without applying physical stress such as sonication to the test object, the biologically-derived soil can be extracted easily and efficiently, and the extracted biologically-derived soil can be detected and quantified.
The living body-derived soil detection and quantification method of the present invention does not need to disassemble the test object and does not apply physical stress to the test object. For example, it is suitable for an endoscope, a laparoscope, and a manipulator, and among them, it is particularly suitable for a manipulator.

The detection and quantification process detects and quantifies dirt derived from living organisms and confirms the cleanliness of the test object. If it is determined that biological contamination is still attached to the test object due to reasons such as inadequate cleaning, the extraction process and the detection and quantification process are performed again after the inspection object is cleaned to detect biological contamination. Quantify.
The inspection object determined that the biologically-derived soil is sufficiently removed can be reused. In addition, since the extraction liquid adheres to the inspection object after the extraction process, it is preferable to rinse the inspection object and reuse it after removing the remaining extraction liquid. The method of rinsing the inspection object is not particularly limited. For example, water such as ion exchange water, distilled water, or RO water is used as the rinsing liquid, and the rinsing liquid is circulated to the inspection object repeatedly. The method of making it contact is mentioned. Further, after the inspection object is rinsed, the inspection object may be treated with a rust preventive lubricant or the like.

The apparatus used in the method for detecting and quantifying biologically-derived soil according to the present invention includes an extraction means that can extract the biologically-derived soil by bringing the extract into fluid contact with the test object, and detection that detects and quantifies the extracted biologically-derived soil. There is no particular limitation as long as it includes a quantitative means.
Hereinafter, an example of the detection and quantification device for biologically derived dirt will be described.

"Detection and quantification device for biologically derived dirt"
FIG. 1 is a schematic configuration diagram showing an example of a living body-derived soil detection and quantification apparatus according to the present invention.
The living body-derived soil detection and quantification apparatus 1 of this example includes an extraction means 10 and a detection and quantification means 20.

<Extraction means 10>
The extraction means 10 is a means for extracting biologically derived dirt by bringing the extraction liquid containing a surfactant into fluid contact with the test object 11.
The extraction means 10 of this example includes a container 12 that accommodates the test object 11 and a supply means 13 that continuously supplies the container 12 with an extract containing a surfactant.

(container)
Although it will not restrict | limit especially if it is a shape which can accommodate the test target object 11 as the container 12, The shape which an extract can contact the inside and the circumference | surroundings of the test target object 11 without a leak is preferable. Examples of such a shape include a box shape, a cylindrical shape, and a bag shape. Further, from the viewpoint of evaporation of the extract and prevention of leakage, the container 12 is preferably one that can accommodate and seal the inspection object 11.

  The material of the container 12 is not particularly limited, but preferably has high temperature resistance, low temperature resistance, solvent resistance, pressure resistance, etc., for example, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyester, AS resin. Thermoplastic resins such as ABS resin, polyethylene terephthalate, (meth) acrylic resin, polyvinyl alcohol, polyvinylidene chloride, polycarbonate, polyamide, polyacetal, polybutylene terephthalate, fluorine resin; phenol resin, melamine resin, urea resin, urethane resin, Thermosetting resins such as epoxy resins and unsaturated polyester resins; Glasses such as quartz glass, soda glass, potash glass, borosilicate glass and lead glass; metals such as aluminum, gold, silver, copper, lead and titanium; stainless steel and duralumin・ Chita Alloy such as alloys; brass, etc. plated metal Tin and the like. Among these, a thermoplastic resin, a thermosetting resin, and glass are preferable from the viewpoint of easy moldability.

The container 12 may have a discharge port 14, for example, as shown in FIG. The discharge port 14 can be attached to and detached from the return pipe 13c.
The container 12 may have a member that enables sealing. Examples of the member that enables sealing include a cap 15 that seals the opening of the container 12 as shown in FIGS. The contact portion between the opening of the container 12 and the cap 15 is preferably sealed with a packing 15a or the like in order to prevent liquid leakage. Further, when the container 12 has a bag shape, for example, as shown in FIGS. 4A and 4B, a chuck 16 may be provided inside the container 12 to enable sealing.
2 to 4 and FIGS. 5 to 9 to be described later, the same components as those in FIG.

Further, when the inspection object 11 accommodated in the container 12 is a manipulator, the container 12 may include an adapter 17 for connecting the supply pipe 13b and the inspection object as shown in FIGS. Good. If the container 12 includes the adapter 17, it is possible to efficiently bring the extract into contact with the inside and the periphery of the inspection object accommodated in the container 12.
Examples of the shape of the adapter 17 include a shape in which a cylinder and a cone are combined as shown in FIG. 5A, and a rectangular parallelepiped shape as shown in FIG. 6A. The inlet is not limited to this as long as it can be connected to the inlet and has a function of reliably feeding liquid into and around the manipulator.

Further, a pore 17 a may be formed on the peripheral surface of the adapter 17. If the pore 17a is formed on the peripheral surface of the adapter 17, as shown in FIG. 5 (b) or FIG. 6 (b), the adapter 17 is attached to the liquid inlet 11b of the manipulator 11a to inject the extract. In this case, the extract is also released from the pores 17a, so that the extract can also be efficiently contacted around the manipulator 11a.
The place where the pores 17a are formed is not limited to the peripheral surface of the adapter 17, and may be the end surface of the adapter 17 as shown in FIGS. 7A and 7B, for example.
Further, as shown in FIGS. 8A and 8B, the adapter 17 may be made of a material or a structure in which a contact portion with the manipulator 11a is expanded according to the pressure of the extract and the extract is leaked. .

  The material of the adapter 17 is not particularly limited, but preferably has high temperature resistance, low temperature resistance, solvent resistance, pressure resistance, etc., for example, natural rubber, nitrile rubber, chloroprene rubber, ethylene propylene rubber, silicone Rubbers such as rubber, butyl rubber, styrene rubber, urethane rubber, fluorine rubber; polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyester, AS resin, ABS resin, polyethylene terephthalate, (meth) acrylic resin, polyvinyl alcohol, polyvinylidene chloride , Polycarbonate, polyamide, polyacetal, polybutylene terephthalate, thermoplastic resin such as fluororesin; thermosetting resin such as phenol resin, melamine resin, urea resin, polyurethane, epoxy resin, unsaturated polyester resin; quartz Glass such as glass, soda glass, potash glass, borosilicate glass and lead glass; metal such as aluminum, gold, silver, copper, lead and titanium; alloy such as stainless steel and duralumin / titanium alloy; plated such as brass and tin Metal etc. are mentioned. From the viewpoint of adhesiveness between the adapter 17 and the manipulator 11a, rubbers, thermoplastic resins, and thermosetting resins are preferable.

(Supply means)
The supply means 13 in this example includes a supply means main body 13a, a supply pipe 13b, and a return pipe 13c.
The supply means main body 13a is not particularly limited as long as the extract can be fed into the container 12, and examples thereof include a pump and a syringe. A pump is preferable from the standpoint that the flow rate of the extract can be easily changed and the extract can be supplied constantly.
Examples of the pump include positive displacement pumps such as reciprocating pumps and rotary pumps; non-displacement pumps such as centrifugal pumps, propeller pumps, and viscous pumps; special pumps such as vortex pumps, jet pumps, submersible pumps, and syringe pumps. . Among these, positive displacement pumps and non-displacement pumps are preferable in view of the amount of liquid fed, and positive displacement pumps are more preferable because the amount of the extract liquid can be easily controlled to be constant.

The supply pipe 13b is a liquid supply pipe for supplying the extract sent out from the supply means main body 13a to the container 12.
The return pipe 13c is a liquid supply pipe for returning the extract discharged from the container 12 to the supply means body 13a.
The container 12 and the supply means main body 13a are connected by a supply pipe 13b and a return pipe 13c.

Although it does not specifically limit as a material of the supply piping 13b and the return piping 13c, It is preferable to have high temperature resistance, low temperature resistance, solvent resistance, pressure | voltage resistance, etc., for example, it illustrated previously in description of the adapter 17 Examples include materials.
The inner diameter and the wall thickness of the supply pipe 13b and the return pipe 13c may be selected according to the diameter, pressure resistance, etc. of the discharge port and the suction port of the supply means body 13a, and are not particularly limited.

(Other components)
For example, as shown in FIG. 9, the extraction unit 10 may include an inclination unit 18, a temperature adjustment unit 19, and the like as necessary.

  The inclination means 18 is means for holding the container 12 at a constant inclination. When the amount of the extract liquid is small, the amount of the extract liquid staying in the container 12 is reduced by the combined use of the inclination means 18, and the circulation efficiency can be improved. The tilting means 18 is not limited as long as it can hold the container 12 at a constant angle.

  The temperature control means 19 is a means for making the temperature of the extract circulated between the container 12 and the supply means 13 constant. As long as the temperature of the extract can be kept constant, the mechanism, structure, installation location, etc. of the temperature control means 19 are not particularly limited. Moreover, you may install a detection fixed_quantity | quantitative_assay device in the temperature-controlled room instead of using the temperature control means 19. FIG.

The extraction means 10 may further include ultrasonic generation means, container decompression means, measuring means such as a thermometer and a flow meter, a cock, and the like (all not shown).
The ultrasonic wave generation unit is a unit for removing bubbles generated in the extract circulated between the container 12 and the supply unit 13. By removing bubbles generated in the extract, the effect of extracting biologically-derived soil is further enhanced. As long as it is possible to remove bubbles generated in the extract, the mechanism and structure of the ultrasonic wave generation means are not particularly limited. The installation location of the ultrasonic wave generation means is not particularly limited as long as the vibration of the ultrasonic wave hardly affects the inspection object such as the manipulator 11a, but it is preferably installed in the middle of the supply pipe or the return pipe.
The ultrasonic irradiation time is preferably within 5 minutes, and preferably within 1 minute.

  The container decompression means is means for decompressing and compressing the container 12. When the material of the container 12 is soft and changes its form due to compression or the like, by compressing the container 12 under reduced pressure, the gap between the container 12 and the inspection object such as the manipulator 11a is reduced and concentrated in and around the inspection object. The extract can be contacted. The container decompression means is not limited as long as the container 12 can be decompressed, and examples thereof include a pump and an aspirator. The decompression is performed by providing a cock at the discharge port of the container 12 and reducing the pressure through the cock at the discharge port, or by providing a cock at the supply pipe 13b and the return pipe 13c and reducing the pressure through the cock of the supply pipe 13b and the return pipe 13c. Or the like.

(Other embodiments)
The extraction means 10 is not limited to that described above. For example, although the extraction means 10 in the illustrated example circulates the extraction liquid between the container 12 and the supply means 13, the extraction liquid need not be circulated.

<Detection quantification means>
The detection quantification means 20 is a means for detecting and quantifying the biologically-derived soil extracted by the extraction means 10.
The detection and quantification means 20 is not particularly limited as long as it can detect and quantitate biologically derived dirt, such as a spectrophotometer, HPLC, GC, GC-MS, etc., an apparatus according to a method for detecting biologically derived dirt, biologically derived dirt. It suffices to have reagents according to the detection method and instruments such as test tubes and pipettes.

<Effect>
The living body-derived dirt detection and quantification device of the present invention described above includes an extraction means for extracting the living body-derived dirt by bringing the extract into fluid contact with the inspection object, so that the inspection object is not decomposed. Without applying physical stress such as sonication that causes a failure to the inspection object, the biologically derived dirt can be extracted easily and efficiently, and the extracted biologically derived dirt can be detected and quantified.
The living body-derived soil detection and quantification apparatus of the present invention does not need to disassemble the inspection object, and does not apply physical stress to the inspection object. For example, it is suitable for an endoscope, a laparoscope, and a manipulator, and among them, it is particularly suitable for a manipulator.

  Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to the following examples.

<Preparation of evaluation device>
Heparin-added sheep blood (manufactured by Nippon Biotest Laboratories Co., Ltd.) and protamine sulfate solution (manufactured by Nacalai Tesque Co., Ltd.) adjusted to a concentration of 1% by mass with ion-exchanged water, heparin-added sheep blood: protamine sulfate solution = The mixture was mixed so as to be 10: 1 (volume ratio) to prepare a simulated protein contamination solution.
5 μL of the simulated protein contaminated solution was applied to the tip of an unused manipulator (EndoWrist (registered trademark) instrument, tip (grip) shape: PRECISE BIPOLAR FORCEPS, manufactured by Intuitive Surgical LLC). Further, 15 μL of the simulated protein contaminated liquid was injected into the housing part from the liquid injection port (main flash port) of the main body part (housing part). Subsequently, it was dried in a constant temperature and humidity chamber (20 ° C., 65% rh.) For 1 hour to obtain an evaluation instrument.
As measured by the BCA method, 3340 μg of protein was contained in a total of 20 μL of the applied simulated protein contamination.

"Example 1"
As the extracting means, the extracting means 10 shown in FIG. 9 was used.
As the container 12, a polyethylene container with a chuck 16 having a silicone outlet 14 detachably attached to the return pipe 13 c was used. As the supply means main body 13a, a pump (manufactured by ASONE Co., Ltd., “compact chemical resistant gear pump GPU-2”) was used. As the supply pipe 13b and the return pipe 13c, a liquid feed pipe made of silicone was used. As the temperature control means 21, a self-coiled heat exchanger (manufactured by ASONE Co., Ltd.) in an oil bath was used.
The container for evaluation was stored in the container 12. A silicone adapter 17 shown in FIG. 5A was attached to the liquid injection port of the evaluation instrument (manipulator), and the inside of the container 12 was sealed by closing the chuck 16 while discharging the air in the container 12. Furthermore, one end of the supply pipe 13b was connected to the adapter 17, and the other end was connected to the pump discharge port 13d of the supply means body 13a.
One end of the return pipe 13c was connected to the discharge port 14 of the container 12, and the other end was connected to the pump suction port 13e of the supply means body 13a.
The container 12 was installed on the tilting means 18, and the return pipe 13 c was immersed in the oil bath of the temperature control means 21.

Using the extraction means 10 described above, the extraction means 10 was operated under the following extraction conditions and fluidly contacted with the evaluation tool while circulating the extract (extraction process).
The viscosity of the extract was measured at a liquid temperature of 20 ° C. using a Canon-Fenske viscometer (manufactured by Shibata Kagaku Co., Ltd., “Viscometer No. 50”).
<Extraction conditions>
Pump flow rate: 130 mL / min Extract liquid volume: 100 mL
Inclination angle: 6 °
Extraction solution: Viscosity of 1% by weight aqueous sodium dodecyl sulfate extract: 1.1 mPa · s
Extraction temperature: 50 ° C
Extraction time: 1, 3, 5, 7, 10, 15, 20, 25, 50 hours

After operating the extraction means 10 until each extraction time, the extract (biologically derived soil (simulated protein) extract) is extracted from the extraction means 10 and the protein is detected and quantified by the BCA method shown below (detection quantification step). . The results are shown in Table 1 and FIG.
Moreover, the state of the manipulator after extraction processing was confirmed by the method shown below. The results are shown in Table 1.

<Protein detection and quantification method by BCA method>
For 1 mL of the simulated protein extract (diluted to 200 μg / mL or less with ion-exchanged water when the amount of protein contained in the simulated protein extract is 200 μg / mL or more), BCA Protein Assay Reagent A (Thermo Fisher) 2 mL of a protein detection solution prepared by mixing 50 mL of Scientific Co., Ltd.) and 1 mL of BCA Protein Assay Reagent B (Thermo Fisher Scientific Co., Ltd.) was added and stirred, and then reacted at 60 ° C. for 30 minutes. Left at 15 ° C. for 5 minutes. The absorbance at 562 nm of the obtained sample was measured with a spectrophotometer (manufactured by PerkinElmer Japan Co., Ltd., “Lambda650S”), and the amount of extracted protein was calculated by substituting it into the formula of the calibration curve for the BCA method created by the following method. Calculated. Moreover, the extraction rate was calculated | required from the following formula.
Extraction rate (%) = extracted protein mass (μg) × 100/3340 (μg)

(Preparation of calibration curve for BCA method)
A bovine serum albumin solution (manufactured by Thermo Fisher Scientific Co., Ltd.) was appropriately diluted with a sodium dodecyl sulfate solution adjusted to a concentration of 1% by mass with ion-exchanged water, and each concentration (0, 0.25, 0.5, 1 5, 50, 100, 150, 200 μg / mL).
Each dilution previously prepared in 2 mL of protein detection solution prepared by mixing 50 mL of BCA Protein Assay Reagent A (manufactured by Thermo Fisher Scientific) and 1 mL of BCA Protein Assay Reagent B (manufactured by Thermo Fisher Scientific) After adding 1 mL of the liquid and stirring, the mixture was reacted at 60 ° C. for 30 minutes, and then allowed to stand at 15 ° C. for 5 minutes to obtain each contact liquid. The absorbance at 562 nm of each of the obtained contact solutions was measured with a spectrophotometer (manufactured by Perkin Elmer Japan, “Lambda650S”), and the absorbance against the protein concentration in the diluted solution was plotted to prepare a calibration curve. The absorbances at protein concentrations of 0, 0.25, 0.5, 1, 5, 50, 100, 150, and 200 μg / mL were obtained linearly.

<Manipulator state>
The manipulator after the extraction treatment was sufficiently rinsed with ion-exchanged water, and then the manipulator was manually operated to visually check whether or not the tip portion operates smoothly. Moreover, after confirmation, the manipulator was disassembled and the state of the internal components was confirmed visually. The state of the manipulator was evaluated according to the following evaluation criteria. “O” is accepted.
○: There is no failure or corrosion such as rust on the parts, and operation is smooth.
Δ: No failure or corrosion such as rust is seen in the parts, but smooth operation is not possible.
×: Corrosion such as failure or rust is seen in the parts, and smooth operation is not possible.

"Example 2"
For comparison with Comparative Examples 1 and 2, an extraction step and a detection quantification step were performed in the same manner as in Example 1. However, the extraction time was 15 hours. Further, the state of the manipulator after the extraction process was confirmed in the same manner as in Example 1. These results are shown in Table 2.

“Comparative Example 1”
The evaluation device is housed in a plastic bag with a chuck, and extracted liquid (a sodium dodecyl sulfate solution adjusted to a concentration of 1% by mass with 0.2N sodium hydroxide) from the liquid injection port of the evaluation device (manipulator). After injecting 100 mL with a syringe, the air in the plastic bag was excluded, and the whole of the evaluation instrument was completely immersed in the extract and sealed.
Next, the entire plastic bag was placed in an ultrasonic pipette washer (Tokyo Rika Kikai Co., Ltd., “AU-175CR”) filled with hot water at 50 ° C., subjected to ultrasonic irradiation for 15 minutes, and then warm water at 50 ° C. And left for 1 hour. This ultrasonic irradiation and leaving were taken as one set, and 12 sets (total of ultrasonic irradiation time and leaving time: 15 hours) were performed (extraction process).
Next, the simulated protein extract in the plastic bag was extracted, and the protein was detected and quantified in the same manner as in Example 1 (detection quantification step). However, a calibration curve prepared as follows was used. Further, the state of the manipulator after the extraction process was confirmed in the same manner as in Example 1. These results are shown in Table 2.

(Preparation of calibration curve for BCA method)
A bovine serum albumin solution (manufactured by Thermo Fisher Scientific Co., Ltd.) was appropriately diluted with a sodium dodecyl sulfate aqueous solution adjusted to a concentration of 1% by mass with 0.2N sodium hydroxide, and each concentration (0, 0.25, 0 .5, 1, 5, 50, 100, 150, 200 μg / mL).
A calibration curve was prepared in the same manner as in Example 1 except that the obtained diluted solution was used. The absorbances at protein concentrations of 0, 0.25, 0.5, 1, 5, 50, 100, 150, and 200 μg / mL were obtained linearly.

“Comparative Example 2”
The extraction process was performed in the same manner as in Comparative Example 1 except that ion-exchanged water was used as the extraction liquid and ultrasonic irradiation was continuously performed for 15 hours.
Next, the simulated protein extract in the plastic bag was extracted, and the protein was detected and quantified in the same manner as in Example 1 (detection quantification step). However, a calibration curve prepared as follows was used. Further, the state of the manipulator after the extraction process was confirmed in the same manner as in Example 1. These results are shown in Table 2.

(Preparation of calibration curve for BCA method)
Bovine serum albumin solution (manufactured by Thermo Fisher Scientific Co., Ltd.) is appropriately diluted with ion-exchanged water, and each concentration (0, 0.25, 0.5, 1, 5, 50, 100, 150, 200 μg / mL) A diluted solution of was obtained.
A calibration curve was prepared in the same manner as in Example 1 except that the obtained diluted solution was used. The absorbances at protein concentrations of 0, 0.25, 0.5, 1, 5, 50, 100, 150, and 200 μg / mL were obtained linearly.

  As apparent from Table 1 and FIG. 10, in the case of Example 1, the extraction rate of the protein was 99.5% or more at the extraction time of 15 hours or more. From this, it was confirmed that a circulation treatment of 15 hours was sufficient for extraction of dirt on the manipulator under the conditions of Example 1. In addition, no manipulator failure was observed even after 50 hours of extraction.

As is apparent from Table 2, in the case of Example 2, the protein extraction rate was 99% or more. In addition, no manipulator failure was observed.
On the other hand, in the case of Comparative Example 1 in which extraction was performed by ultrasonic irradiation, the extraction rate was similar to that in Example 2, but black and white precipitates were generated inside the manipulator after the extraction process. Wire breakage was also observed.
In the case of the comparative example 2 which extracted by ultrasonic irradiation using ion-exchange water as an extract, the extraction rate was low. Moreover, the tip of the manipulator after the extraction process did not operate smoothly.

  According to the detection and quantification method and detection and quantification apparatus for biologically-derived soils of the present invention, the physical stress that causes a failure can be detected without disassembling the inspection object having a precise and complicated structure such as a manipulator. It is possible to easily and efficiently extract and quantitate biologically derived dirt without subjecting it to sewage. Therefore, the living body-derived soil detection and quantification method and the detection and quantification apparatus of the present invention of the present invention are used to evaluate the cleanliness of inspection objects such as manipulators reused in medical institutions and the like, and performance evaluation of medical washing machines and the like. It can be particularly preferably used.

DESCRIPTION OF SYMBOLS 1 Detection fixed_quantity | quantitative_assay device 10 Extraction means 11 Test object 11a Manipulator 11b Liquid inlet 12 Container 13 Supply means 13a Supply means main body 13b Supply piping 13c Return piping 13d Pump discharge port 13e Pump suction port 14 Discharge port 15 Cap 15a Packing 16 Chuck 17 Adapter 17a Pore 18 Tilting means 19 Temperature control means 20 Detection and quantification means

Claims (7)

A method for detecting and quantifying biologically-derived soil adhering to an inspection object,
An extraction step of fluidly contacting an extraction liquid containing a surfactant with the test object to extract biologically derived dirt; and
A detection and quantification process for detecting and quantifying the extracted biologically-derived soil;
A method for detecting and quantifying soil derived from a living body.   The method for detecting and quantifying biologically-derived soil according to claim 1, wherein the extract is circulated and repeatedly brought into fluid contact with the test object.   The method for detecting and quantifying biologically derived dirt according to claim 1 or 2, wherein the test object is an instrument that has been washed after use.   The method for detecting and quantifying biologically-derived soil according to any one of claims 1 to 3, wherein the extract has a viscosity of 0.1 to 500 mPa · s.   The inspection object is a manipulator having a liquid injection port, an adapter is attached to the liquid injection port, the extraction liquid is injected, and the extraction liquid is fluidly contacted with the manipulator. The method for detection and quantification of biologically-derived soil as described in 1. A device for detecting and quantifying living body-derived dirt adhered to an inspection object,
An extraction means for fluidly contacting an extract containing a surfactant with the test object to extract biologically derived dirt, and a detection quantitative means for detecting and quantifying the extracted biologically-derived dirt;
The said extraction means is a detection-quantification apparatus of biological origin dirt provided with the container which accommodates the said test object, and the supply means which supplies the said extract continuously to the said container.   The biological fluid according to claim 6, wherein the extract discharged from the container is returned to the supply means, and the extract is circulated between the container and the supply means and repeatedly fluidly contacts the test object. Dirt detection and quantification device.

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