Method for collecting saliva, saliva collection component, method for determining the presence or absence of bleeding, and method for determining the progression of periodontal disease.

Abstract

Provided is a determination method with which it is possible to find a saliva collection member achieving simple and noninvasive collection, and by simply subjecting the collection member after saliva collection to centrifugal separation without any special treatment, occult blood in saliva can be determined and inflammatory-inducing lactoferrin polypeptide concentration in the saliva can be measured, enabling disease state classification of a periodontal disease in humans and animals.　In this invention, saliva is noninvasively collected by selecting and using a nonwoven cloth capable of quantitatively collecting saliva. The nonwoven cloth after the collection is set to a filtration device to apply centrifugal separation. A blood component remaining on the nonwoven cloth after the centrifugal separation is visually observed to detect occult blood in the saliva. Moreover, by also measuring the I-Lf concentration in the liquid phase specimen after the centrifugal separation, it is possible to classify the condition into the appropriate one of periodontal disease states.

Description

[Technical Field]

[0001] The present invention relates to a method for collecting saliva, a saliva collection member, a method for determining the presence or absence of bleeding, and a method for determining the progression of periodontal disease. [Background technology]

[0002] In humans and pets such as dogs, periodontal disease is a serious lifestyle-related disease that can trigger a wide range of illnesses, including endocarditis and diabetes (Non-patent Literature 1, Relationship between Periodontal Disease and Lifestyle-Related Diseases, edited by the Human 8020 Promotion Foundation, 2005). Therefore, in humans, regular checkups involve measuring periodontal pockets using a probe (probe) as shown in Figure 1, as well as examining bleeding and redness of periodontal tissue. However, examining the affected area with a probe is often painful.

[0003] In recent years, non-invasive tests, such as measuring occult blood in saliva due to bleeding from periodontal tissue or measuring blood components in saliva, have become more common. However, these tests still have drawbacks, such as the need for dilution and the time required for measurement, and are not yet widely adopted.

[0004] On the other hand, for animals such as dogs, X-ray examinations and periodontal pocket measurements cannot be performed without general anesthesia, which places a great burden on the pet itself and results in high medical costs due to the need for hospitalization (Non-patent document 2, Survey on the Actual State of Medical Fees and Owner Awareness of Pet Owners for Domestic Animals (Dogs and Cats) (FY2015), edited by the Japan Veterinary Medical Association. 2015).

[0005] Furthermore, while bacterial culture and genetic testing using saliva are beginning to be performed, a simple and accessible saliva testing method has not yet been established. Periodontal disease is diagnosed by observing whether the position of periodontal tissues has receded, by measuring the depth of periodontal pockets with a probe, and by measuring the presence or absence of bleeding and pus due to periodontal disease. The diagnosis is made using the Oral Hygiene Index (OHI) based on the evaluation of the plaque index (Non-Patent Literature 3 J. Amer. Dent.). Ass. 61: 172- 179, 1960.), PLI (Plaque Index) (Non-patent document 4 Acta. Odont. Scand. 22: 121-135, 1964.) and the GI (Gingival Index) for evaluating gingival inflammation (Non-patent Literature 5 Acta. Odont. Scand. 21: 533-551, In dental practice, assessment methods such as those described in 1963 are used.

[0006] The methods described above are expensive and place a heavy burden on the affected animals, so there is a need for a non-invasive examination method that is effective, inexpensive, and can be used to assess the pathology of periodontal disease. Furthermore, these diagnostic methods are time-consuming because they involve all teeth, and require skilled techniques. In addition, saliva-based diagnostic methods are beginning to be introduced to test for pain in inflamed areas of gum tissue caused by periodontal disease.

[0007] The following technologies have been implemented as methods for determining non-invasiveness: Saliva occult blood detection kit (Patent Document 1: Japanese Patent No. 4590581, Non-Patent Document 6: Japanese Journal of Periodontology 43 (4): 416423, 2001). Fecal occult blood detection kit (Non-patent document 7: Research on the standardization of saliva testing, 8020 Promotion Foundation Designated Research Project Report, 2012). Method for measuring lactate dehydrogenase (Patent Document 2: Japanese Patent Publication No. 2010-130924, Non-Patent Document 8: Japanese Journal of Periodontology 44 (3): 261-272) 2002).

[0008] However, with immunochromatographic saliva occult blood detection kits, it is necessary to spit out 1 mL or more of saliva into a paper cup using sugar-free gum, etc., and then dilute it five times with water or rinse with 3 mL of water for 10 seconds to minimize the influence of substances that inhibit membrane development such as mucin (Non-patent Literature 9, Perioscreen "Sunstar" Package Insert, 2017).

[0009] On the other hand, while salivary occult blood testing using fecal occult blood detection kits involves collecting and measuring saliva, similar to immunochromatographic kits, it must be performed at an external laboratory equipped with expensive automated analyzers (Non-patent Literature 10, Fecal Occult Blood Kit OC-Hemodia Auto III 'Eiken' Package Insert, 2021). Other methods for detecting and identifying periodontal disease-causing bacteria in saliva include conventional bacterial testing methods and the Polymerase Chain Reaction (PCR) method, a genetic test, which are performed at external testing facilities equipped with specialized equipment (Non-patent Literature 11 GCCIRCLE.141:2012-2016.2012. Patent Literature 3: Japanese Patent Application Publication No. 2017-85944). Because early-stage periodontal disease often shows few noticeable symptoms, these tests are intended for patients with moderate to severe periodontal disease who already exhibit noticeable symptoms such as clear bleeding when using a probe or a growing concern about bad breath.

[0010] On the other hand, in animals such as dogs, periodontal disease examinations are generally performed under general anesthesia (Non-patent Literature 13: On Periodontal Disease in Dogs (Diagnosis Method) | Pocket Pets Doctors https: / / pocketpetsdoctors.com / ?p=134 2021). We have already reported that "inflammatory lactoferrin polypeptide (I-Lf)" appears in the saliva of patients with periodontal disease (Non-patent Literature 12 Mol. Immunol. 44:1498-1508. 2007. Patent Literature 4: Japanese Patent No. 4029988), and demonstrated its effectiveness in detecting patients with early-stage periodontal disease without the use of probes using immunosorbent assay (ELISA).

[0011] Similar to I-Lf measurement, salivary occult blood measurement, which determines bleeding in saliva, is performed to detect periodontal disease early, as described above. Among these, there have been reports on the measurement of salivary occult blood using nonwoven fabric (Patent Document 6, Japanese Patent No. 2860660), in which hemolytic components and various drugs are immersed in nonwoven fabric, then dried, and then attached to a support such as polyvinyl chloride and the color is determined. More specifically, in Patent Document 6, an indicator that changes color and is oxidized in the presence of blood-replenishing molecules present in the blood, an oxidizing agent effective in oxidizing the indicator, a buffer that maintains the pH in the range of 4 to 7, an activator to increase sensitivity, and saponin are impregnated into an absorbent carrier selected from paper, cellulose, chemical fibers, synthetic resin woven fabric, and nonwoven fabric.

[0012] As a nonwoven fabric, the nonwoven fabric described in Patent Document 7 is known. This nonwoven fabric has a basis weight of 30-80 g / m² according to JIS P8124:1998. 2 Sheet density 0.01~0.05 g / cm³ 3 The compression work by the KES method is 0.85-5.0 gf·cm / cm 2 It is a nonwoven fabric with a wet tensile strength of 8 N / 100 mm or more. However, its intended use is cooking paper, and no other uses are disclosed. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] Patent No. 4590581 [Patent Document 2] Japanese Patent Publication No. 2010-130924 [Patent Document 3] Japanese Patent Publication No. 2017-85944. [Patent Document 4] Patent No. 4029988 [Patent Document 5] Patent No. 4627263 [Patent Document 6] Japanese Patent No. 2860660 [Patent Document 7] Japanese Patent No. 6259365 [Non-Patent Document]

[0014] [Non-Patent Document 1] Relationship between periodontal disease and lifestyle diseases. Edited by the Foundation for the Promotion of Human 8020. 2005. [Non-Patent Document 2] Survey on the actual state of medical fees for household pets (dogs and cats) and the awareness of pet owners (FY2017), edited by the Japan Veterinary Medical Association. 2015. (http: / / nichiju.lin.gr.jp / small / ryokin.html) [Non-Patent Document 3] J. Amer. Dent. Ass. 61: 172 - 179, 1960. [Non-Patent Document 4] Acta. Odont. Scand. 22: 121 - 135, 1964. [Non-Patent Document 5] Acta. Odont. Scand., 21: 533 - 551, 1963. [Non-Patent Document 6] Nihishushu Shiki 43 (4): 416 - 423, 2001. [Non-Patent Document 7] Research on the standardization of saliva tests. Report of the designated research project of the Foundation for the Promotion of 8020. 2012. [Non-Patent Document 8] Nihishushu Shiki 44 (3): 261 - 272, 2002. [Non-Patent Document 9] Attachments of Perioscreen "Sunstar". 2017. [Non-Patent Document 10] GCCIRCLE.141:2012 - 2016.2012. [Non-Patent Document 11] Mol. Immunol. 44:1498 - 1508. 2007. [Non-Patent Document 12] Fecal occult blood test kit OC-Hemodia Auto III 'Eiken' package insert, 2021. [Non-Patent Document 13] About Periodontal Disease in Dogs (Diagnosis Methods) | Pocket Pets Doctors https: / / pocketpetsdoctors.com / ?p=134 2021. [Non-Patent Document 14] J. Clin. Pathol 31: 139-143 1978. [Non-Patent Document 15] Clin. Chim. Acta. 136. 95-104. 1984. [Overview of the project] [Problems that the invention aims to solve]

[0015] In this invention, in order to quantify I-Lf in saliva, there are challenges in selecting a nonwoven fabric that can quantitatively collect saliva and establishing a measurement method. Furthermore, the collection method must be non-invasive and not cause pain to patients or animals such as pets. In particular, in pets, general anesthesia is currently required to observe the oral cavity, so there is a challenge in establishing a collection method that does not require anesthesia.

[0016] For non-invasive saliva collection methods, we envision using a nonwoven fabric that is safe for use in living organisms and is also being considered as a saliva collection material capable of measuring salivary occult blood. We will use a nonwoven fabric that is readily available and does not require special processing, and we will investigate a method that allows for simple and rapid determination of salivary occult blood using the nonwoven fabric after saliva absorption. As chemical testing methods, the orthotolidine method and the guaiac method are routinely used for fecal occult blood tests and are also used in some cases for salivary occult blood tests.

[0017] Furthermore, immunological methods utilizing antigen-antibody reactions are used for lower gastrointestinal and colonic bleeding, as well as for salivary occult blood (Non-patent Literature 9: Perioscreen "Sunstar" Package Insert, 2017).

[0018] Patent Document 6 describes a method in which hemolytic components and various drugs are immersed in a nonwoven fabric, which is then dried and attached to a support such as polyvinyl chloride for determination by color. However, this method requires complicated procedures and materials. Furthermore, although the details of the nonwoven fabric are not disclosed, when commercially available nonwoven fabrics are used, the amount of saliva necessary for determination is not absorbed, making it impossible to perform stable determinations. The inventors further investigated the cause of the inability to perform stable determinations and found that the cause lies in the variability of the absorption amount. In this invention, the inventors searched for a nonwoven fabric that could control the absorption amount to be constant and found that the nonwoven fabric described in Patent Document 7 has a large saliva absorption amount and low variability. In addition, it is also used for filtering highly viscous edible oils and has a high ability to retain impurities. Therefore, the inventors found that it is possible to detect latent blood donation in saliva by attaching the saliva-absorbing nonwoven fabric to a centrifugal filtration sterilization filter or a centrifugal ultrafiltration filter.

[0019] Currently reported measurement methods for salivary occult blood, I-Lf, and causative bacteria do not include a method for classifying the pathology of periodontal disease based on saliva samples. Therefore, we aim to solve this problem by establishing a diagnostic method that enables the classification of periodontal disease states using both I-Lf concentration and salivary occult blood as indicators, which are effective in detecting periodontal disease and are also indicators in saliva. The present invention aims to provide a method for easily collecting saliva for periodontal disease examination in humans, and for easily collecting saliva in animals such as dogs and cats without anesthesia. The present invention aims to provide a saliva collection method and saliva collection component that are simple, non-invasive, require no processing, and can be used without complicated procedures or materials, enabling the determination of occult blood in saliva and the stable measurement of the inflammatory lacloferrin polypeptide concentration in saliva with little variation, simply by centrifugation. It also aims to provide a method for determining the presence or absence of bleeding and a method for determining the progression of periodontal disease.

[0020] An object of the present invention is to provide a method for simply and quickly determining the progression of periodontal disease by using saliva as a specimen, and a saliva collection method that can be performed non-invasively without causing pain by a probe in humans and without requiring anesthesia in pets or the like. Another object of the present invention is to provide a saliva collection method by which a person can determine periodontal disease by himself / herself, and a pet owner can determine periodontal disease in pets without anesthesia. Means for Solving the Problems of the Invention

[0021] The present invention relates to a saliva collection method for periodontal disease examination, which is a saliva collection method for collecting saliva by absorbing the saliva of a human or an animal such as a dog or a cat with a saliva collection member made of a non-woven fabric capable of quantitatively absorbing saliva.

[0022] The non-woven fabric preferably has a basis weight of 30 to 80 g / m according to JIS P8124:1998 2 , a sheet density of 0.01 to 0.05 g / cm 3 , a compression work amount of 0.85 to 5.0 gf·cm / cm according to the KES method 2 , and a wet tensile strength of 8 N / 100 mm or more. The present invention is a method for determining the presence or absence of bleeding by observing the blood cell components retained in the saliva collection member after centrifugation, after collecting saliva by the saliva collection method, by attaching the saliva collection member containing saliva to a centrifugal filtration filter and centrifuging. The present invention is a method for determining the progression of periodontal disease by measuring both the pro-inflammatory lactoferrin polypeptide and the occult blood measurement in the liquid phase obtained by the centrifugation described above. The present invention is a saliva collection member used for a saliva collection method for periodontal disease examination, which is a saliva collection member made of a non-woven fabric capable of quantitatively absorbing saliva. The non-woven fabric preferably has a basis weight of 30 to 80 g / m according to JIS P8124:1998 2 , a sheet density of 0.01 to 0.05 g / cm 3 , a compression work amount of 0.85 to 5.0 gf·cm / cm according to the KES method 2 , and a wet tensile strength of 8 N / 100 mm or more. [Effect of the Invention]

[0023] The effects of the present invention will be explained along with the knowledge gained in the process of developing it. The present inventors have discovered that the nonwoven fabric described in Patent Document 7 has the following characteristics necessary for determining periodontal disease using I-Lf concentration and salivary occult blood as indicators: (1) a large amount of saliva absorbed per unit volume, and (2) extremely little variation in the amount of saliva absorbed per unit volume (and consequently, the overall amount of saliva absorbed). Based on the discovery of these characteristics, the inventors have developed a saliva collection member for use in determining periodontal disease using I-Lf concentration.

[0024] For saliva collection, a nonwoven fabric (product name "Lead") manufactured by Oji KinoCross Co., Ltd. using the airlaid method is used. This nonwoven fabric has a higher wet tensile strength than nonwoven fabrics produced by other methods, is less prone to tearing during physical processing such as centrifugation, and has the characteristic of minimizing paper fragment shedding. In immunoturbidimetry, I-Lf measurement requires 2-3 repeated measurements to improve the reliability of the measurement results. Therefore, a sample volume of 40 μL is required for two measurements. Furthermore, considering the possibility of dilution and re-measurement using immunoturbidimetry, it is necessary to be able to collect a sample volume of 60 μL or more. In this invention, the nonwoven fabric is cut to a volume of 84 mm³ that can be attached to the reservoir of a centrifugal filtration filter, and it was confirmed that it can quantitatively absorb 100 μL of highly viscous saliva, which is one of the body fluids, as 99.5 + 0.5 μL (Table 1). [Table 1]

[0025] As shown in Patent Document 7, although it is made from wood pulp, any cooking paper with performance equivalent to that of the cooking paper, such as natural fibers like cotton and rayon, or synthetic fibers like polyester and polypropylene, that can absorb even highly viscous oils, can be used. Therefore, the nonwoven fabric made from the aforementioned material is adjusted to match the amount of saliva to be collected, and the amount of saliva required for the test is collected non-invasively, simply, and quickly.

[0026] The collected saliva is placed in a centrifugal filter or similar filtration device equipped with a membrane filter with a pore size of 0.2 μm or 0.45 μm to remove the influence of bacteria, or an ultrafiltration membrane matched to the molecular weight of the saliva components to be measured. The sample is then centrifuged at 1,000 to 3,000 G (centrifugal force) for 5 to 15 minutes, conforming to the specifications of the filtration device. I-Lf, an indicator for periodontal disease, contained in the liquid phase after centrifugation can be measured by antigen-antibody reactions, enzyme reactions, or chemical color reactions. Meanwhile, occult blood remaining in the saliva collection material could be visually confirmed on the nonwoven fabric after centrifugation by checking for bleeding. Previously, processing such as adding colorants to the nonwoven fabric was necessary, but this invention demonstrates a method that does not require such processing, allowing for simple determination by simply attaching the fabric to a sterilized filtration filter and centrifuging it. The method for attaching the nonwoven fabric to the centrifugal filtration apparatus is as shown in Figure 2. By folding it into a V-shape or U-shape and attaching it to the apparatus, blood cell components accumulate on the nonwoven fabric that comes into contact with the bottom of the filtration filter, making visual confirmation possible. It was found that the amount of saliva absorbed differed by more than twice as much between this nonwoven fabric and nonwoven fabrics produced by other methods, and the variation in the amount of saliva retained also differed by more than 100 times, making it impossible to stably collect a sufficient amount of saliva sample for measurement. Therefore, it was demonstrated that the use of this nonwoven fabric is an extremely effective saliva collection material in this measurement method.

[0027] Furthermore, to quantitatively confirm even minute amounts of bleeding, we have established a method that allows for the quantitative analysis of blood components contained in the nonwoven fabric after centrifugation by dispersing them in a buffer solution suitable for each measurement method and measuring their absorbance using international standard methods such as the cyanmethemoglobin method (Non-patent Literature 14 J. Clin. Pathol. 31. 139-143. 1978.) or the Triton / NaOH method (Non-patent Literature 15 Clin. Chim. Acta. 136. 95-104. 1984.).

[0028] According to the invention, a method for collecting saliva for periodontal disease examination can be provided that is simple in humans and can also be performed simply without anesthesia in animals such as dogs and cats. According to the present invention, it is possible to provide a saliva collection method and saliva collection component that are simple, non-invasive, can be used without any processing, and can determine the presence of occult blood in saliva and measure the concentration of inflammatory lacloferrin polypeptide in saliva with little variation and stability simply by centrifugation, without requiring complicated work or materials, as well as a method for determining the presence or absence of bleeding and a method for determining the progression of periodontal disease. Table 1 shows the results of the study on the saliva absorption capacity of the nonwoven fabric in question. [Brief explanation of the drawing]

[0029] [Figure 1] Flowchart of saliva sample processing method and measurement process [Figure 2] Example of calibration curve creation using immunoturbidimetry [Figure 3] I-Lf concentration in saliva samples from animals (dogs) suffering from periodontal disease, depending on the disease state. [Figure 4] I-Lf concentration in saliva samples from periodontal disease patients, depending on the disease state. [Figure 5] Sensitivity test for observing occult blood in saliva using the nonwoven fabric used in the present invention [Figure 6] External photograph showing a bleeding specimen from a dog suffering from periodontal disease. [Figure 7] Schematic diagram of periodontal disease diagnosis using a probe [Modes for carrying out the invention]

[0030] This invention simultaneously measures I-Lf, a very early periodontal disease detection marker contained in the saliva of humans and animals suffering from periodontal disease, and the presence or absence of bleeding, which increases in the saliva due to bleeding associated with the progression of periodontal disease, to determine the progression of periodontal disease. In this test, the saliva-absorbing material used was a nonwoven fabric (product name "Lead") from Oji Kinocross Co., Ltd., pre-cut to absorb 100 μL each of human and canine saliva, with a volume of 84 mm². 3The sample was cut to the specified size, and saliva was collected from the patient and the animal. Note that the size is not limited to the above size; any size that allows for the absorption of 100 μL of sample is acceptable. From this perspective, 80-90mm 3 Preferably, 82-86 mm 3 This is preferable.

[0031] The saliva-soaked nonwoven fabric was then folded and placed in a commercially available centrifugal filter loaded with a pore size 0.2 μm membrane filter, and centrifuged at 2,000 G for 5 minutes. After centrifugation, the nonwoven fabric was placed on a white acrylic plate or similar surface, and the presence or absence of blood cell components was visually observed and determined.

[0032] Next, the filtrate after centrifugation was analyzed for I-Lf using an affinity antibody prepared from anti-I-Lf rabbit serum, employing immunoturbidimetric analysis for rapid measurement. For the analysis, Tris-HCl buffer (pH 7.2) containing 4.5% polyethylene glycol 8000 was used as the reaction buffer, and 120 μL was dispensed into each well of a microplate (MICROLON 762070, greirer bio-one) and incubated at 37°C. Then, the i-Lf affinity antibody was adjusted to 1 mg / mL with Tris-HCl buffer, 15 μL was dispensed into each well, and mixed with a plate mixer. After mixing, 20 μL of the saliva sample after centrifugation was added and mixed again, and the reaction was incubated at 37°C for 8 minutes. After the reaction was complete, the plate was mixed again with a plate mixer to resuspend the immune complex, and the absorbance was measured at 540 nm to determine the salivary I-Lf concentration. Furthermore, in cases where occult blood was detected in saliva and was thought to exceed the measurement range, the sample was diluted with Tris-HCl buffer and measured again.

[0033] In canine saliva, the value was 8.89 ± 5.25 μg / ml for mild to moderate cases, but higher at 21.21 ± 11.26 μg / ml in the group judged to be severe. However, there was considerable variability between the severe and mild to moderate groups, making it difficult to classify symptoms based solely on I-Lf measurement. On the other hand, the measurement time was significantly reduced from approximately 2 hours for the conventional immunosorbent assay (ELISA) method to 5-10 minutes from the start of measurement to obtaining results. On the other hand, in human saliva samples, the glycemic index (GI) was high at 62.2±10.3 μg / ml in patients classified as severely ill based on their GI value, but it was lower in patients with milder symptoms (31.0±8.3 μg / ml) and mild symptoms (8.4±2.0 μg / ml).

[0034] These results suggest that changes in I-Lf concentration due to periodontal disease are more sensitive in humans than in dogs, making classification easier. Furthermore, it is presumed that in humans, various self-management measures such as brushing teeth, using mouthwash and interdental brushes are taken to maintain oral hygiene, thus suppressing plaque and tartar buildup and reducing individual differences in disease state. In contrast, in dogs, there are differences in management methods and awareness regarding maintaining oral hygiene, such as brushing teeth, and it is presumed that there are large individual differences in oral hygiene, resulting in greater variability in I-Lf values.

[0035] In measuring occult blood in saliva, non-hemolytic components such as red blood cells have a relatively large particle size, while hemolytic components such as hemoglobin are difficult to observe visually. Therefore, we investigated the measurement sensitivity of hemoglobin using commercially available hemoglobin (SIGMA, Cat. No. H7379-5G) dissolved in phosphate physiological buffer and serially diluted from 1 mg / mL to 0.01 μg / mL. Each concentration of hemoglobin solution was placed in a nonwoven fabric, and the mixture was centrifuged at 2,000 G for 15 minutes using a commercially available centrifuge filter loaded with a 0.45 μm membrane filter. After separating the liquid sample, it was removed and observed visually. As a result, visual confirmation was possible from 1 μg / mL to 1 mg / mL using this measurement method. On the other hand, the measurement sensitivity of commercially available immunochromatographic human saliva occult blood detection kits is 2 μg / mL to less than 500 μg / mL (Non-patent document, Perioscreen "Sunstar" package insert, 2017), which suggests that it is approximately the same as the method of the present invention.

[0036] The results are shown in Figure 5. Based on the results described above, we observed occult blood in canine saliva. We found that as the condition of periodontal disease worsened, the detection rate of occult blood in saliva increased from not being detected in samples with mild periodontal disease to 88% in moderate cases and 100% in severe cases. Furthermore, the detection rate of shed epithelial tissue in the oral cavity also increased as the condition worsened, from not being detected in mild cases to 12% in moderate cases and 80% in severe cases.

[0037] While observing occult blood in saliva is effective in checking the progression of periodontal disease, it is difficult to detect periodontal disease in mild cases using only occult blood observation. Combining this with I-Lf suggests that the progression of periodontal disease can be more efficiently and accurately assessed. The results are shown in Table 2. Specifically, Table 2 shows the measurement results for occult blood and shed epithelium in the saliva of dogs infected with periodontal disease. The bleeding samples are shown in Figure 6. [Table 2]

[0038] By simply centrifuging a nonwoven fabric without any processing, it became possible to determine the presence of occult blood in saliva. This allowed for the simultaneous measurement of two parameters: occult blood in saliva and salivary I-Lf concentration. This enabled more accurate assessment of periodontal disease and dramatically reduced the time required for diagnosis. Furthermore, this method can be applied to measuring parameters other than I-Lf that are effective in diagnosing periodontal disease. The nonwoven fabric after saliva collection contains concentrated bacteria that did not pass through the filtration membrane, so it can be suspended in a small amount of sterile buffer solution and used as a concentrated sample for genetic testing and bacterial culture. In addition, the liquid phase can be used for measuring various substances such as immunoglobulins, other proteins, minerals, and enzymes. Therefore, this method has a wide range of applications, including primary screening for periodontal disease in general health checkups for humans and animals, as well as oral hygiene and health examinations.

[0039] In pets such as dogs, detailed examinations require general anesthesia, making it more difficult to diagnose periodontal disease compared to humans, and often leading to delayed detection. As a result, periodontal disease is often discovered only after it has become severe. The present invention enables the detection of periodontal disease using a non-invasive examination method, allowing for early treatment. Furthermore, it reduces the burden on pets caused by general anesthesia and contributes to reducing the high cost of pet medical care.

Claims

1. This is a method for collecting saliva for periodontal disease examination, in which saliva is collected by allowing a saliva collection member, made of a nonwoven fabric capable of absorbing saliva, to absorb human or animal saliva. The aforementioned nonwoven fabric has a size of 80-90 mm ３ A saliva collection method in which, for every 100 μL of saliva added, 100 μL ± 0.5 μL of saliva is absorbed.

2. A method for determining the presence or absence of bleeding, comprising collecting saliva using the saliva collection method according to claim 1, attaching the saliva-containing saliva collection member to a centrifugal filter and centrifuging it, and observing the blood cell components retained in the saliva collection member after centrifugation.

3. A method for determining the progression of periodontal disease by measuring the inflammatory lactoferrin polypeptide in the liquid phase obtained by centrifugation according to claim 2, in conjunction with measuring occult blood.

4. This is a saliva collection component used in a saliva collection method for periodontal disease examination, and is made of a nonwoven fabric that can absorb saliva. The aforementioned nonwoven fabric has a size of 80-90 mm ３ The saliva collection member has a saliva absorption rate of 100 μL ± 0.5 μL for every 100 μL of saliva added.

5. This is a method for collecting saliva for periodontal disease examination, in which saliva is collected by allowing a saliva collection member, made of a nonwoven fabric capable of absorbing saliva, to absorb human or animal saliva. The standard deviation of saliva absorption per unit volume is ±0.65 μL / 168 mm ３ The following is the method for collecting saliva.

6. The aforementioned nonwoven fabric has a basis weight of 30-80 g / m² according to JIS P8124:1998. ２ , seat close degree 0.01~0.05g / cm ３ The compression work by the KES method is 0.85 to 5.0 gf·cm / cm ２ The saliva collection method according to claim 1, wherein the nonwoven fabric has a wet tensile strength of 8 N / 100 mm or more.

7. This is a saliva collection component used in a saliva collection method for periodontal disease examination, and is made of a nonwoven fabric that can absorb saliva. The standard deviation of saliva absorption per unit volume is ±0.65 μL / 168 mm ３ The following are components for collecting saliva.

8. The aforementioned nonwoven fabric has a basis weight of 30-80 g / m² according to JIS P8124:1998. ２ , seat close Degree: 0.01 to 0.05 g / cm ３ and the compression work amount by the KES method is 0.85 to 5.0 gf·cm / cm ２ The saliva collection member according to claim 4, which is a non-woven fabric having a wet tensile strength of 8 N / 100 mm or more.

9. A method for collecting saliva for periodontal disease examination, which involves collecting saliva by allowing human or animal saliva to be absorbed into a saliva collection member made of nonwoven fabric with a standard deviation of saliva absorption less than 64.2 μL.

10. A method for collecting saliva for periodontal disease examination, which involves collecting saliva by allowing human or animal saliva to be absorbed into a saliva collection member made of nonwoven fabric with a standard deviation of saliva absorption less than 0.65 μL.

11. This is a saliva collection component used in a saliva collection method for periodontal disease examination, and it has a standard deviation of saliva absorption amount. A saliva collection component made of nonwoven fabric with a difference of less than 64.2 μL.

12. A saliva collection component used in a saliva collection method for periodontal disease examination, wherein the standard deviation of the amount of saliva absorbed is 0.65 μL or less.

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