Saliva collection method, saliva collection unit, method for determining presence or absence of bleeding, and method for determining progression state of periodontal disease

Abstract

The present invention discovers a simple and non-interventional saliva collection member, and provides a saliva collection member that can be used without any processing. The present invention relates to a determination method capable of determining occult blood in saliva, measuring the inflammatory lactoferrin-polypeptide concentration in saliva, and classifying periodontal diseases of humans and animals, only by centrifugally separating a collection member from which saliva has been collected. By selectively using the non-woven fabric capable of quantitatively collecting the saliva, the saliva can be collected in a non-intervention manner. The collected non-woven fabric is arranged in a filter tool for centrifugal separation, blood components remaining on the centrifuged non-woven fabric are visually observed, and saliva occult blood is detected. In addition, the concentration of I-Lf in the centrifuged liquid-phase sample is measured together, so that periodontal diseases can be classified according to the conditions of the periodontal diseases.

Description

Technical Field

[0001] The present invention relates to a method for collecting saliva, a saliva collection component, a method for determining the presence or absence of bleeding, and a method for determining the development status of periodontal disease. Background Art

[0002] For humans and pets such as dogs, periodontal disease is a major lifestyle disease that induces various diseases such as endocarditis and diabetes (Non-Patent Document 1: Relationship between periodontal disease and lifestyle diseases. Edited by the Foundation for the Promotion of Human 8020. 2005.). Therefore, for humans, through regular management, examinations such as measurement of periodontal pockets, bleeding, and redness of periodontal tissues are performed using instruments such as Figure 1 the probe shown. However, when using a probe to examine the affected area, pain is often accompanied.

[0003] In recent years, as a painless examination, that is, a non-invasive examination, determination of occult blood in saliva accompanied by bleeding from periodontal tissues and determination of blood components contained in saliva have been started, but there are problems such as time-consuming dilution during measurement and time-consuming measurement, and thus it has not been popularized.

[0004] On the other hand, for animals such as dogs, X-ray examination and measurement of periodontal pockets cannot be performed without general anesthesia, so there are problems of a large burden on the pet itself, the need for hospitalization, and high medical costs (Non-Patent Document 2: Survey of actual medical fees and awareness of breeders of household pets (dogs and cats) (2015 fiscal year), edited by the Japanese Veterinary Medical Association. 2015.).

[0005] In addition, although culture of pathogenic bacteria and gene examination have also been started as examinations using saliva, a simple saliva examination method that anyone can perform has not been established.

[0006] Periodontal disease is determined by observing whether the position of the periodontal tissue has not decreased, measuring the depth of the periodontal pocket using a probe, and further measuring the presence or absence of bleeding or pus caused by periodontal disease. Determination is performed at the dental site using determination methods such as OHI (Oral Hygiene Index) based on evaluation of the plaque index (Non-Patent Document 3: J. Amer. Dent. Ass. 61: 172-179, 1960.), PLI (Plaque Index) (Non-Patent Document 4: Acta. Odont. Scand. 22: 121-135, 1964.), and GI (Gingival Index) for evaluating gingival inflammation (Non-Patent Document 5: Acta. Odont. Scand. 21: 533-551, 1963.).

[0007] The inspection cost of the above method is high and the burden on the affected animals is also large. Therefore, there is a need to provide a non-invasive inspection method that can effectively and inexpensively determine the condition of periodontal disease.

[0008] In addition, since these determination methods are carried out for all the teeth of the object, they are time-consuming determination methods, determination methods that require skilled techniques, and a determination method using saliva has been introduced for the examination of the pain at the inflammatory site of the gingival tissue caused by periodontal disease.

[0009] As a method introduced as a non-invasive determination method, the following techniques are implemented.

[0010] Saliva occult blood detection kit (Patent Document 1: Japanese Patent No. 4590581, Non-Patent Document 6: Japanese Journal of Periodontology 43(4):416 - 423, 2001).

[0011] Fecal occult blood detection kit (Non-Patent Document 7: Research Report on the Standardization of Saliva Examination, Project Report of the 8020 Promotion Foundation for Specified Research. 2012).

[0012] Method for measuring lactate dehydrogenase (Patent Document 2: Japanese Unexamined Patent Application Publication No. 2010 - 130924, Non-Patent Document 8: Japanese Journal of Periodontology 44(3):261 - 272, 2002).

[0013] However, in the saliva occult blood detection kit using immunochromatography, in order to spit out more than 1 mL of saliva into a paper cup or the like using sugar-free chewing gum or the like and suppress the influence of substances that hinder the development of mucin etc. on the membrane, it is necessary to dilute it 5 times with water, or it is necessary to rinse the mouth with 3 mL of water for 10 seconds and use it as the spit-out liquid (Non-Patent Document 9: Attachment Document of PeriodoScreen "Sunstar", 2017.).

[0014] On the other hand, in the saliva occult blood examination using a fecal occult blood detection kit, although saliva is collected and measured in the same way as in the kit based on immunochromatography, it must be carried out at an external examination institution equipped with an expensive automatic analysis device (Non-Patent Document 10: Attachment Document of Fecal Occult Blood Kit OC-Hemodia Auto III 'Eiken', 2021.).

[0015] The method using the usual bacterial examination method for detecting periodontal disease pathogens contained in saliva through other detections and the gene examination, that is, the polymerase chain reaction (PCR) method, also needs to be carried out at an external examination institution equipped with dedicated equipment (Non-Patent Document 11: GCCIRCLE.141:2012 - 2016. 2012. Patent Document 3: Japanese Unexamined Patent Application Publication No. 2017 - 85944.).

[0016] Since it is difficult to have self-awareness of early periodontal disease, these examinations are targeted at patients with moderate or more severe conditions. Compared with patients in the initial stage, there are already cases where bleeding is clearly visible when using a probe, or self-awareness symptoms such as bad breath have begun to be noticed.

[0017] On the other hand, for animals such as dogs, general anesthesia is usually implemented during the examination of periodontal disease (Non-Patent Document 13: About Periodontal Disease in Dogs (Diagnostic Method) | Pocket Pets Doctors (POCKET PETSDOCTORS) https: / / pocketpetsdoctors.com / ?p=1342021.).

[0018] We have reported that "inflammatory lactoferrin-polypeptide (I-Lf)" appears in the saliva of periodontal disease patients (Non-Patent Document 12: Mol. Immunol. 44: 1498-1508. 2007. Patent Document 4: Japanese Patent No. 4029988 Gazette), and it has been shown that early-stage periodontal disease patients can be effectively detected by the immunoenzymatic antibody (ELISA) method without using a probe.

[0019] Similar to the measurement of I-Lf, in order to detect early periodontal disease, the measurement of salivary occult blood for determining bleeding in saliva is performed as described above. Among them, the measurement of salivary occult blood using non-woven fabric has also been reported (Patent Document 6: Japanese Patent No. 2860660 Gazette). After impregnating the hemolytic component and various agents in the non-woven fabric and drying it in an air-dried manner, it is attached to a support such as polyvinyl chloride and judged by colorimetry. More specifically, in Patent Document 6, an indicator that is oxidized with a color change due to the presence of the blood complement molecule family in blood, an oxidizing agent effective for oxidizing the indicator, a buffer for maintaining the pH in the range of 4 to 7, an activator for improving sensitivity, and saponin are impregnated in an absorbent carrier selected from paper, cellulose, chemical fiber, synthetic resin woven fabric, and non-woven fabric.

[0020] As the non-woven fabric, the non-woven fabric described in Patent Document 7 is known. The unit area weight of this non-woven fabric based on JIS P8124: 1998 is 30 to 80 g / m 2 、and the sheet density is 0.01 to 0.05 g / cm 3 、the compression work based on the KES method is 0.85 to 5.0 gf·cm / cm 2 、and the wet tensile strength is 8 N / 100 mm or more. However, its use is for cooking paper, and other uses are not disclosed.

[0021] Prior Art Documents

[0022] Patent Documents

[0023] Patent Document 1: Japanese Patent Publication No. 4590581

[0024] Patent Document 2: Japanese Unexamined Patent Application Publication No. 2010-130924

[0025] Patent Document 3: Japanese Unexamined Patent Application Publication No. 2017-85944

[0026] Patent Document 4: Japanese Patent Publication No. 4029988

[0027] Patent Document 5: Japanese Patent Publication No. 4627263

[0028] Patent Document 6: Japanese Patent Publication No. 2860660

[0029] Patent Document 7: Japanese Patent Publication No. 6259365

[0030] Non-Patent Documents

[0031] Non-Patent Document 1: Relationship between Periodontal Disease and Lifestyle Diseases. Edited by the Foundation for the Promotion of Human 8020. 2005.

[0032] Non-Patent Document 2: Survey Results on the Actual Status of Medical Fees for Domestically Raised Animals (Dogs and Cats) and the Awareness of Breeders (Fiscal Year 2015), Edited by the Japan Veterinary Medical Association. 2015. (http: / / nichiju.lin.gr.jp / small / ryokin.html)

[0033] Non-Patent Document 3: J.Amer.Dent.Ass. 61:172-179, 1960.

[0034] Non-Patent Document 4: Acta.Odont.Scand. 22:121-135, 1964.

[0035] Non-Patent Document 5: Acta.Odont.Scand., 21:533-551, 1963.

[0036] Non-Patent Document 6: Japanese Journal of Periodontology 43(4):416-423, 2001.

[0037] Non-Patent Document 7: Research Report on the Standardization of Saliva Tests, Report of the Specified Research Project of the Foundation for the Promotion of 8020. 2012.

[0038] Non-Patent Document 8: Japanese Journal of Periodontology 44(3):261-272, 2002.

[0039] Non-Patent Document 9: Package Insert of "Sunstar" Perioscreen, 2017.

[0040] Non-patent Document 10: GCCIRCLE.141: 2012 - 2016. 2012.

[0041] Non-patent Document 11: Mol. Immunol. 44: 1498 - 1508. 2007.

[0042] Non-patent Document 12: Instruction Manual of Fecal Occult Blood Kit OC - Hemodia Auto III 'Eiken', 2021.

[0043] Non-patent Document 13: About Periodontal Disease in Dogs (Diagnostic Method) | Pocket Pets Doctors (POCKETPETSDOCTORS) https: / / pocketpetsdoctors.com / ?p = 134 2021.

[0044] Non-patent Document 14: J. Clin. Pathol 31: 139 - 143 1978.

[0045] Non-patent Document 15: Clin. Chim. Acta. 136. 95 - 104. 1984. Summary of the Invention

[0046] Problems to be Solved by the Invention

[0047] The problem of the present invention is to establish a method for quantitatively collecting saliva using a non-woven fabric that can quantitatively collect saliva and is selected for use at the time of collection in order to quantify I-Lf in saliva. In addition, a non-invasive method that does not cause pain to patients or diseased animals such as pets during collection is required. Especially for pets, etc., general anesthesia is currently required when observing the oral cavity, so there is a problem of establishing a collection method that does not require anesthesia.

[0048] Assume that a non-invasive saliva collection method is a method of collecting by holding or spitting out a collection component contained in the oral cavity, etc., and study the selection of non-woven fabric as a saliva collection component that can safely measure fecal occult blood in saliva even when used on living organisms. Non-woven fabric is a component that is generally easily available without special processing methods, and study a method for simply and quickly determining fecal occult blood in saliva using the non-woven fabric after saliva absorption. As chemical examination methods, the orthotolidine method and the guaiac method are also used for part of the fecal occult blood examination in saliva as routine examinations.

[0049] In addition, for lower digestive tract, large intestine bleeding, and fecal occult blood in saliva (Non-patent Document 9: Instruction Manual of Periodo Screen 'Sunstar', 2017.), immunological methods using antigen-antibody reactions are adopted.

[0050] In Patent Document 6, a hemolytic component and various medicaments are impregnated into a nonwoven fabric and then dried by an air-drying method, and then attached to a support such as polyvinyl chloride for determination by colorimetry. However, this requires complicated operations and materials. In addition, although the details of the nonwoven fabric are not disclosed, in the case of using a commercially available nonwoven fabric, stable determination cannot be performed because the required amount of saliva is not absorbed. The present inventors further investigated the reason why stable determination could not be performed, and as a result, it was found that the reason lies in the deviation of the absorption amount. In the present invention, a nonwoven fabric capable of controlling the absorption amount to be constant was investigated, and as a result, it was found that the nonwoven fabric described in Patent Document 7 has a large saliva absorption amount and a small deviation. In addition, since it is also used for filtering highly viscous edible oils and the like, the ability to retain inclusions is also high. Therefore, the following insight was obtained: by mounting the nonwoven fabric that has absorbed saliva on a centrifugal filtration sterilization filter or a centrifugal ultrafiltration filter, occult blood in saliva can be detected.

[0051] In the currently reported measurement methods for saliva occult blood determination, I-Lf, and pathogenic bacteria, etc., no inspection method for classifying the condition of periodontal disease using a saliva sample has been established. Therefore, taking both the I-Lf concentration and saliva occult blood, which are effective as determination indicators in saliva for detecting periodontal disease, as indicators, a determination method capable of classifying the periodontal condition was established in order to solve the problem.

[0052] An object of the present invention is to provide a saliva collection method that can simply collect saliva for periodontal disease examination for humans, and can also simply collect saliva for animals such as dogs and cats without anesthesia.

[0053] An object of the present invention is to provide a saliva collection method, a saliva collection component, a method for determining the presence or absence of bleeding, and a method for determining the development state of periodontal disease that are simple, non-invasive, can be used without any processing, do not require complicated operations and materials, and can determine occult blood in saliva only by centrifugation and can stably measure the concentration of proinflammatory lactoferrin-polypeptide in saliva with a small deviation.

[0054] An object of the present invention is to provide a method for simply and quickly determining the development state of periodontal disease by using saliva as a sample, and a saliva collection method that is non-invasive, can be performed on humans without the pain caused by a probe, does not require anesthesia-based treatment for pets, etc., and can be used by the person himself / herself to determine periodontal disease for humans and by the owner to determine periodontal disease for animals.

[0055] Means for Solving the Problem

[0056] The present invention relates to a method for collecting saliva for periodontal disease examination, which collects saliva by making a saliva collection component composed of non-woven fabric capable of quantitatively absorbing saliva absorb the saliva of humans or animals such as dogs and cats.

[0057] The non-woven fabric is preferably a non-woven fabric having a basis weight of 30 to 80 g / m based on JIS P8124:1998 2 , a sheet density of 0.01 to 0.05 g / cm 3 , a compression work based on the KES method of 0.85 to 5.0 gf·cm / cm 2 , and a wet tensile strength of 8 N / 100 mm or more.

[0058] The present invention relates to a method for determining the presence or absence of bleeding, which collects saliva by the saliva collection method, and then mounts a saliva collection component containing saliva on a centrifugal filter for centrifugation, and observes the blood cell components remaining in the saliva collection component after centrifugation.

[0059] The present invention relates to a method for determining the development status of periodontal disease, which determines the development status of periodontal disease by simultaneously measuring pro-inflammatory lactoferrin-polypeptide and occult blood in the liquid phase obtained by the above centrifugation.

[0060] The present invention relates to a saliva collection component, which is a saliva collection component used in a saliva collection method for periodontal disease examination and is composed of non-woven fabric capable of quantitatively absorbing saliva.

[0061] The non-woven fabric is preferably a non-woven fabric having a basis weight of 30 to 80 g / m based on JIS P8124:1998 2 , a sheet density of 0.01 to 0.05 g / cm 3 , a compression work based on the KES method of 0.85 to 5.0 gf·cm / cm 2 , and a wet tensile strength of 8 N / 100 mm or more.

[0062] Advantages of the Invention

[0063] The advantages of the present invention are described together with the insights obtained when the present invention was completed.

[0064] The inventor of the present invention found that the non-woven fabric described in Patent Document 7 has the characteristics of (1) a large saliva absorption amount per unit volume required for determining periodontal disease using I-Lf concentration and saliva occult blood as indicators, and (2) very little deviation in the saliva absorption amount per unit volume (and thus the deviation in the overall saliva absorption amount). Based on the discovery of these characteristics, a use for a saliva collection component for determining periodontal disease based on I-Lf concentration was developed, and the present invention was completed accordingly.

[0065] For the collection of saliva, non-woven fabric (trade name "LAID") manufactured by OJI KINOCLOTH Co., Ltd. made by the airlaid method is used. Compared with non-woven fabrics made by other methods, this non-woven fabric has stronger wet tensile strength and has the property that it is not easily damaged even during physical treatments such as centrifugation and has less shedding of paper pieces.

[0066] In the determination of I-Lf by immunoturbidimetry, in order to improve the reliability of the measured value, it is necessary to measure repeatedly 2 to 3 times. Therefore, a sample volume of 40 μL is required for 2 measurements. In addition, considering the case of dilution and re-measurement in immunoturbidimetry, it is necessary to be able to collect more than 60 μL. In the present invention, using the said non-woven fabric, it is cut in such a way that it can be installed in the reservoir of a centrifugal filter and the volume reaches 84 m 3 and it was confirmed that 99.5 + 0.5 μL of 100 μL of highly viscous saliva in body fluid can be quantitatively absorbed (Table 1).

[0067] [Table 1]

[0068]

[0069] Saliva addition amount: 84 m 3 100 μL, 168 m 3 200 μL

[0070] Number of repeated tests: 11 times

[0071] Company A; Non-woven fabric made by OJI KINOCLOTH, Non-woven fabric made by Company B, Non-woven fabric made by Company C

[0072] Downward: Absorption amount + standard deviation

[0073] As shown in Patent Document 7, although it is made from wood pulp, whether it is natural fibers such as cotton and rayon or synthetic fibers such as polyester and polypropylene, as long as it has the same performance as the said cooking paper that can absorb highly viscous oil, it can be used. Therefore, it is characterized in that the non-woven fabric made from the said raw materials is adjusted according to the amount of saliva collected, and the saliva required for examination is simply and quickly collected non-invasively.

[0074] In addition, in order to eliminate the influence of miscellaneous bacteria and the like, the collected saliva is placed in a centrifugal filter equipped with a membrane filter having a pore size of 0.2 μm or 0.45 μm or an ultrafiltration membrane selected according to the molecular weight of the saliva component as a measurement index, or a centrifugal filtration device having a similar function, and centrifuged at 1,000 to 3,000 G (centrifugal force) for about 5 to 15 minutes according to the specifications of the filtration device. I-Lf, which is a determination index for periodontal disease, contained in the liquid phase after centrifugation can be measured by antigen-antibody reaction, enzyme reaction, chemical color reaction, etc. On the other hand, after centrifugation, the presence or absence of bleeding on the non-woven fabric is visually confirmed for the occult blood in the saliva remaining in the saliva collection material. So far, it has been necessary to perform processing such as adding a color developing agent to the non-woven fabric, but in the present invention, a method is shown in which determination can be simply performed only by centrifugation without these processes by installing it in a filtration sterilization filter. The method of installing the non-woven fabric in the centrifugal filtration device is as Figure 2 shown, and is characterized in that it is installed in the device by folding it into a V shape or a U shape, and the blood cell components are aggregated on the non-woven fabric in contact with the bottom of the filter, and can be visually confirmed. It has been found that for the non-woven fabric and the non-woven fabric made by other methods, the difference in saliva retention is more than 2 times and the difference in the deviation of the retention amount is more than 100 times with respect to the amount of saliva absorbed, and it is impossible to stably collect a sufficient saliva sample required for measurement. Therefore, in this measurement method, it is shown that the use of the non-woven fabric is an extremely effective saliva collection component.

[0075] In addition, in order to further quantitatively confirm trace bleeding, a method has been established that can also be performed as follows: The blood components contained in the centrifuged non-woven fabric are dispersed in a buffer suitable for each measurement method, and the absorbance is measured and quantified using an international standard method, that is, cyanmethemoglobin (Non-Patent Document 14: J. Clin. Pathol. 31. 139-143. 1978.), Triton / NaOH method (Non-Patent Document 15: Clin. Chim. Acta. 136. 95-104. 1984.), etc.

[0076] According to the invention, a saliva collection method can be provided, which can simply collect saliva for periodontal disease examination for humans, and can also be simply collected without anesthesia even for animals such as dogs and cats.

[0077] According to the present invention, a saliva collection method, a saliva collection component, a method for determining the presence or absence of bleeding, and a method for determining the development state of periodontal disease can be provided, which are simple, non-invasive, can be used without any processing, do not require complicated operations and materials, can determine the occult blood in saliva only by centrifugation, and can stably measure the concentration of pro-inflammatory lactoferrin-polypeptide in saliva with a small deviation.

[0078] The research results of the saliva absorption amount of the non-woven fabric are shown in Table 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 It is a flowchart of the processing method of the saliva sample and the process to the measurement.

[0080] Figure 2 It is an example of making a standard curve using immunoturbidimetry.

[0081] Figure 3 It is the I-Lf concentration under the influence of the disease condition of the saliva sample of an affected animal (dog) suffering from periodontal disease.

[0082] Figure 4 It is the I-Lf concentration under the influence of the disease condition of the saliva sample of a patient suffering from periodontal disease.

[0083] Figure 5 It is an observation sensitivity test for occult blood in saliva using the non-woven fabric used in the present invention.

[0084] Figure 6 It is a photograph showing the appearance of a bleeding sample of a dog suffering from periodontal disease.

[0085] Figure 7 It is a schematic diagram of the diagnosis of periodontal disease using a probe. DETAILED DESCRIPTION OF THE INVENTION

[0086] The present invention simultaneously measures I-Lf, which is a marker for determining periodontal disease at a very early stage, contained in the saliva of a person or animal suffering from periodontal disease, and whether the bleeding in the saliva increases due to bleeding accompanying the development of periodontal disease, and determines the development status of periodontal disease.

[0087] In this experiment, as the saliva absorption member, the non-woven fabric of OJI KINOCLOTH Co., Ltd. (product name: "LAID") was previously cut into a size of 84 mm 3 to absorb 100 μL of human and dog saliva respectively, and saliva was collected from patients and affected animals. In addition, it is not limited to the above size, as long as it is a size that can absorb 100 μL of the sample. From this point of view, it is preferably 80 to 90 mm 3 and more preferably 82 to 86 mm 3 .

[0088] And, the non-woven fabric that has absorbed saliva is bent and placed in a commercially available centrifugal filter filled with a membrane filter having a pore size of 0.2 μm, and centrifuged at 2,000 G for 5 minutes. Then, the centrifuged non-woven fabric is placed on an acrylic plate with a white background, etc., and judged by visually observing the presence or absence of blood cell components.

[0089] Next, for the filtrate after centrifugation, I-Lf was measured by immunoturbidimetry using an affinity antibody prepared from anti-I-Lf rabbit serum, which enables measurement in a short time. The measurement used Tris-HCl buffer (pH 7.2) containing 4.5% polyethylene glycol 8000 as the reaction buffer. 120 μL was dispensed into each well of a microplate (MICROLON 762070, greirer bio-one) and incubated at 37°C. Next, the i-Lf affinity antibody was adjusted to 1 mg / mL with Tris-HCl buffer, 15 μL was dispensed into each well, and after mixing with a plate mixer, 20 μL of the centrifuged saliva sample was added, mixed again, and reacted in a constant temperature bath at 37°C for 8 minutes. After the reaction ended, in order to resuspend the immune complex, it was mixed again with a plate mixer, and the absorbance was measured at 540 nm to determine the I-Lf concentration in the saliva. It should be noted that for samples considered to have occult blood in saliva exceeding the measurement range, they were diluted with Tris-HCl buffer and measured again.

[0090] For canine saliva, the value was 8.89 ± 5.25 μg / ml in the mild to moderate range, while it was a higher value of 21.21 ± 11.26 μg / ml in the group determined to be severe. However, the deviation in each of the severe and mild to moderate groups was large, and it was difficult to apply the results to the classification of their respective symptoms based solely on the measurement of I-Lf. On the other hand, compared with the measurement time of about 2 hours for the previously performed immunoenzymatic antibody method (ELISA method), the measurement time from the start of measurement to obtaining the result was significantly shortened to 5 - 10 minutes.

[0091] On the other hand, for human saliva samples, the value was as high as 62.2 ± 10.3 μg / ml in the saliva of patients determined to be severe based on the GI value, while the saliva of patients with milder symptoms, such as moderate patients' saliva (31.0 ± 8.3 μg / ml) and mild patients' saliva (8.4 ± 2.0 μg / ml), was lower.

[0092] This result suggests that for humans, the concentration change of I-Lf caused by periodontal disease changes more acutely compared to dogs and is easier to classify compared to dogs. In addition, for humans, because various self-management measures such as brushing, mouthwash, and interdental brushes are used to maintain the oral hygiene environment, it is speculated that the deposition of dental plaque and calculus is inhibited, and thus the individual differences caused by the disease become smaller. In contrast, in the case of dogs, it is speculated that there are differences in management methods and awareness such as brushing regarding the maintenance of the oral hygiene environment, and the individual differences in the oral hygiene environment are large, resulting in a large deviation in the I-Lf value.

[0093] In the determination of occult blood in saliva, red blood cells as non-hemolytic components have a relatively large particle size. In contrast, hemolytic components such as hemoglobin are difficult to observe visually. Therefore, commercially available hemoglobin (SIGMA, Cat. No. H7379 - 5G) was dissolved in phosphate physiological buffer, and solutions diluted from the 1 mg / mL stage to 0.01 μg / mL were used to study the detection sensitivity of hemoglobin. Hemoglobin solutions of each concentration were incorporated into non-woven fabric, and a commercially available centrifugal filter equipped with a 0.45 μm membrane filter was centrifuged at 2,000 G for 15 minutes. After separating the liquid sample, it was taken out and observed visually. As a result, in this assay method, 1 μg / mL to 1 mg / mL could be confirmed visually. On the other hand, the detection sensitivity of a commercially available human saliva occult blood detection kit based on immunochromatography is 2 μg / mL to 500 (<) μg / mL (Non-patent literature: Perioscreen "Sunstar" attached document, 2017.), suggesting that it is roughly equivalent to the method of the present invention.

[0094] The results are shown in Figure 5 。

[0095] Based on the above results, occult blood in canine saliva was observed. As the periodontal disease worsened, the detection rate of occult blood in saliva was not detected in samples with mild periodontal disease, increased to 88% in moderate cases, and increased to 100% in severe cases. In addition, for exfoliated epithelial tissues in the oral cavity, as the disease worsened, it was not detected in mild cases either, but the detection rate increased to 12% in moderate cases and to 80% in severe cases.

[0096] To confirm the development status of periodontal disease, the observation of occult blood in saliva is effective. However, for mild samples, it is difficult to detect periodontal disease only through the observation of occult blood, suggesting that the combination with I-Lf can more effectively and accurately judge the development status of periodontal disease.

[0097] The results are shown in Table 2. That is, Table 2 shows the results of the determination of occult blood and exfoliated epithelium in saliva samples of dogs suffering from periodontal disease. In addition, the bleeding samples are as Figure 6 shown.

[0098] [Table 2]

[0099]

[0100] Mild: The number of samples is 6. Moderate: The number of samples is 11. Severe: The number of samples is 12

[0101] Without any processing, the determination of occult blood in saliva is completed only by centrifuging the non-woven fabric. Thus, two items, namely the determination of occult blood in saliva and the concentration of I-Lf in saliva, can be measured simultaneously, enabling a more accurate determination of the condition of periodontal disease and a dramatic reduction in the determination time.

[0102] In addition, in this method, in addition to I-Lf, it can also be applied to the measurement of items effective for the determination of periodontal disease. Since the non-woven fabric after saliva collection contains bacteria that have not passed through the filter membrane in a concentrated state, it can be used as a concentrated test sample for genetic testing and bacterial culture by suspending it in a small amount of sterilized buffer solution. In addition, the liquid phase part can be used for various measurements such as immunoglobulins, other proteins, inorganic substances, and enzymes. Therefore, the present invention is a method that can be applied to a wide range of fields such as primary screening for periodontal disease, oral hygiene, and health diagnosis in routine physical examinations of humans and animals.

[0103] In pets such as dogs, general anesthesia is required for detailed examinations. Therefore, compared with humans, it is difficult to determine periodontal disease and there is a tendency to detect it relatively late. As a result, many cases are discovered only after they have become severe. With a non-invasive inspection method such as the present invention, periodontal disease can be detected, enabling early treatment to begin. In addition, while reducing the burden on pets caused by general anesthesia, it also helps to reduce high medical costs.

Claims

1. A method for collecting saliva for periodontal disease examination, which collects saliva by allowing a saliva collection component made of non-woven fabric capable of quantitatively absorbing saliva to absorb the saliva of humans or animals such as dogs and cats.

2. The saliva collection method according to claim 1, wherein, The non-woven fabric has a basis weight of 30 to 80 g / m² based on JIS P8124:1998 2 , a sheet density of 0.01 to 0.05 g / cm³ 3 , a compression work based on the KES method of 0.85 to 5.0 gf·cm / cm² 2 , and a wet tensile strength of 8 N / 100 mm or more 3. A method for determining the presence or absence of bleeding, which, after collecting saliva by the saliva collection method described in claim 1, mounts the saliva collection component containing saliva on a centrifugal filter for centrifugation, and observes the blood cell components remaining in the saliva collection component after centrifugation.

4. A method for determining the development status of periodontal disease, which determines the development status of periodontal disease by simultaneously measuring pro-inflammatory lactoferrin-polypeptide and occult blood measurement in the liquid phase obtained by centrifugation in claim 3.

5. A saliva collection component, which is a saliva collection component used in the saliva collection method for periodontal disease examination, and is made of non-woven fabric capable of quantitatively absorbing saliva.

6. The saliva collection component according to claim 5, wherein, The non-woven fabric has a basis weight of 30 to 80 g / m² based on JIS P8124:1998 2 , a sheet density of 0.01 to 0.05 g / cm³ 3 , a compression work based on the KES method of 0.85 to 5.0 gf·cm / cm² 2 , and a wet tensile strength of 8 N / 100 mm or more

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