CN116218949A - Preservation method of periodontitis biomembrane saliva inoculant - Google Patents

Abstract

The invention discloses a preservation method of periodontitis biomembrane saliva inoculant, which comprises the following steps: 24 hours before saliva collection, the subjects did not perform any oral hygiene, and no food or drink was taken for the first 2 hours. Each subject discharges 10ml of unstimulated whole saliva into a 15ml sterile freezer tube using a funnel and inserts into ice. 1ml of mineral oil was added to the super clean bench, taking care not to blow and suck, the mineral oil being in the upper layer of saliva. The freezing tube is placed into a 2.5L round bottom vertical anaerobic culture bag, an outer packing bag of the 2.5L anaerobic gas production bag is torn open, the anaerobic gas production bag is placed into the freezing tube, two circles of freezing tubes are folded along the sealing part of the culture bag, the freezing tubes are fixed by clamps, and the freezing tubes are placed into a refrigerator at 4 ℃ for preservation, and are used for subsequent saliva inoculum evaluation and periodontitis biomembrane inoculation culture. The method prolongs the survival time of bacteria in the saliva inoculant, particularly anaerobic bacteria, effectively prolongs the saliva preservation time, and is suitable for preserving periodontitis biological membrane saliva inoculant.

Description

Preservation method of periodontitis biomembrane saliva inoculant

Technical field:

the invention relates to the technical field of microorganisms, in particular to a preservation method of periodontitis biomembrane saliva inoculant.

The background technology is as follows:

periodontitis is one of the most common diseases in humans, currently affecting about 5.38 hundred million people worldwide. Periodontitis is caused by oral biofilm imbalance, and thus, it is very necessary to build a suitable biofilm model. The inoculum is a key factor in establishing a model of oral biofilms, which are not usually the result of specific strain action in real oral environments, but rather are formed by the mutual cooperation, competition and metabolism of different microorganisms, so that the multi-species model has advantages over single species, and currently known researches of periodontitis biofilms often use saliva or subgingival bacterial plaque as inoculum. The survival biomass of saliva or subgingival plaque as an inoculum, as well as the survival nature of the surviving species, in particular of the specific flora associated with periodontitis diseases, plays a critical role in the success of constructing periodontitis biofilms.

The largest difference between the saliva flora in the disease state and the saliva flora in the health state is that the saliva flora contains more anaerobic bacteria, saliva is treated within 30 minutes after sampling when a periodontitis biological film model is built, the biological film is cultured, and the treatment time before the test is shorter. There are different kinds of saliva preserving solutions in the market, but the preserving solution can only reduce the degradation of biological genetic materials in saliva, and can not lead the bacterial colony in saliva to survive for a long time.

To meet the current need for constructing periodontitis biofilms, there is a need for a method that preserves the saliva inoculum of periodontitis biofilms by reserving more time for the test treatment saliva without affecting the viable biomass and the viable bacterial species in the saliva.

The invention comprises the following steps:

the invention aims to overcome the defect that saliva treatment time is short and saliva preservation time for inoculation is short when a periodontitis biological film model is built, and provides a method for preserving periodontitis biological film saliva inoculant.

The technical scheme adopted for solving the technical problems is as follows:

a method of preserving a periodontitis biofilm saliva inoculum comprising the steps of:

the periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each volunteer discharged 10ml of unstimulated whole saliva directly into a 15ml sterile freezer using a funnel, and after the end, the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. Placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip;

after all samples were collected, the anaerobic bags were kept in a refrigerator at 4 ℃ for subsequent saliva inoculum evaluation and inoculation periodontitis biofilm culture.

Further, the funnel and 15ml freezer tube were steam autoclaved at 121 ℃ for 15 minutes before use.

Further, the mineral oil was autoclaved at 121 ℃ for 15 minutes before use, followed by sub-packaging at a super clean bench.

Further, periodontitis subjects discharged 10ml of saliva into 15ml sterile freezer tubes using a funnel in a sterile room, quickly unscrewed the cap, and put on ice.

Further, the sterile freezer tube was opened at a clean bench, 1ml of sterilized and packaged mineral oil was slowly added, note that the mineral oil was not mixed with saliva, and the cap was screwed on the upper layer of saliva.

Further, placing the sterile freezing tube filled with saliva after oil sealing into a 2.5L round bottom vertical anaerobic culture bag, tearing an outer packing bag of the 2.5L anaerobic gas production bag, placing into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip.

The beneficial effects are that:

1. the method prolongs the survival time of bacteria in saliva inoculums, particularly anaerobic bacteria after collecting saliva, effectively prolongs the time of saliva treatment when constructing periodontitis biological membranes, and is suitable for preserving periodontitis biological membrane saliva inoculums. Meanwhile, saliva preserved by the method is not influenced by mineral oil, and the subsequent requirement of inoculum evaluation can be met.

2. The method establishes a preservation method of periodontitis biomembrane saliva inoculant, and fills up the gap of the method.

3. The materials adopted by the invention are low in price and easy to obtain, and the method is simple and efficient.

The drawings in the specification:

FIG. 1 is a diagram showing the result of agarose gel electrophoresis of the amplified product of the saliva sample DNA 16S universal primer of the present invention

Wherein: 1 is the PCR amplification product band of example 1 (0 hour of storage), 2 is the PCR amplification product band of example 2 (4 ℃ C. For 4 hours), 3 is the PCR amplification product band of example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours), and 4 is the PCR amplification product band of comparative example 1 (fresh saliva is now collected);

FIG. 2 is a diagram showing the result of agarose gel electrophoresis of saliva sample DNA Propionibacterium amplification product preserved in the present invention

Wherein: 1 is the PCR amplification product band of example 1 (0 hour of storage), 2 is the PCR amplification product band of example 2 (4 ℃ C. For 4 hours), 3 is the PCR amplification product band of example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours), and 4 is the PCR amplification product band of comparative example 1 (fresh saliva is now collected);

FIG. 3 is a diagram showing agarose gel electrophoresis results of a saliva sample DNA Fusobacterium amplification product stored in the present invention

Wherein: 1 is the PCR amplification product band of example 1 (0 hour of storage), 2 is the PCR amplification product band of example 2 (4 ℃ C. For 4 hours), 3 is the PCR amplification product band of example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours), and 4 is the PCR amplification product band of comparative example 1 (fresh saliva is now collected);

FIG. 4 is a diagram showing the result of agarose gel electrophoresis of the amplified product of the DNA treponema of the saliva sample preserved in the present invention

Wherein: 1 is the PCR amplification product band of example 1 (0 hour of storage), 2 is the PCR amplification product band of example 2 (4 ℃ C. For 4 hours), 3 is the PCR amplification product band of example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours), and 4 is the PCR amplification product band of comparative example 1 (fresh saliva is now collected);

FIG. 5 shows the result of agarose gel electrophoresis of saliva sample DNA Porphyromonas amplification product preserved in the present invention

Wherein: 1 is the PCR amplification product band of example 1 (0 hour of storage), 2 is the PCR amplification product band of example 2 (4 ℃ C. For 4 hours), 3 is the PCR amplification product band of example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours), and 4 is the PCR amplification product band of comparative example 1 (fresh saliva is now collected);

FIG. 6 shows agarose gel electrophoresis results of saliva sample DNA Tannerella amplified products preserved in the present invention

Wherein: 1 is the PCR amplification product band of example 1 (0 hour of storage), 2 is the PCR amplification product band of example 2 (4 ℃ C. For 4 hours), 3 is the PCR amplification product band of example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours), and 4 is the PCR amplification product band of comparative example 1 (fresh saliva is now collected);

FIG. 7A general bar graph of real-time fluorescent quantitative PCR Ct values

The specific embodiment is as follows:

the present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.

The raw materials used in the invention are conventional commercial products unless specified otherwise, the methods used in the invention are conventional methods in the art unless specified otherwise, and the mass of each substance used in the invention is conventional.

1. Qualitative PCR detection of surviving bacteria in saliva under different storage conditions

Example 1 (preservation for 0 hours)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer using a funnel, and after completion the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. The freezing tube is placed into a 2.5L round bottom vertical anaerobic culture bag, an outer packing bag of the 2.5L anaerobic gas production bag is torn, the 2.5L anaerobic gas production bag is placed into the freezing tube, two circles of freezing tube are folded along the sealing position of the culture bag, and the freezing tube is fixed by a clamp.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. 3ml of the supernatant was taken in a sterile 10ml Ep tube, 10. Mu.l of PMA solution was added, mixed by blowing and sucking, incubated in the dark for 5min, and then exposed to intense light of a 650w halogen lamp (placed at 25cm from the sample) for 2min, the whole procedure being carried out on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. PCR was performed using the DNA as a template and the 6 sets of primers of Table 1, the amplification system is shown in Table 2, and the amplification procedure is shown in Table 3.

TABLE 1 primer sequences of genus Bacillus and annealing temperatures

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TABLE 2 PCR amplification System

TABLE 3 PCR amplification procedure

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, the annealing temperature of the primers of the Proteus, the Fusobacterium, the Porphyromonas, the Treponema and the Tannerella are 58 ℃, and the number of amplification cycles is 35.

Example 2 (4 ℃ C. For 4 hours)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each volunteer discharged 10ml of unstimulated whole saliva directly into a 15ml sterile freezer using a funnel, and after the end, the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. Placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip; the anaerobic culture bag is put into a refrigerator with the temperature of 4 ℃ for 4 hours.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. 3ml of the supernatant was pipetted into a sterile 10ml Ep tube, 10. Mu.l of PMA solution was added, mixed by blowing and pipetting, incubated in the dark for 5min, and then exposed to intense light from a 650w halogen lamp (placed 25cm from the sample) for 2min, the whole procedure being carried out on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. PCR was performed using the DNA as a template and the 6 sets of primers of Table 1, the amplification system is shown in Table 2, and the amplification procedure is shown in Table 3.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, the annealing temperature of the primers of the Proteus, the Fusobacterium, the Porphyromonas, the Treponema and the Tannerella are 58 ℃, and the number of amplification cycles is 35.

Example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer using a funnel, and after completion the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. Placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip; the anaerobic culture bag is put into a refrigerator with the temperature of 4 ℃ for 4 hours.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. The supernatant was dispensed with 50% glycerol, 750. Mu.l of supernatant and 750. Mu.l of glycerol were added to each glycerol tube, and the mixture was air-sucked and mixed, and saliva-glycerol samples were stored in a-80℃refrigerator for 24 hours. After 24 hours, 6ml of saliva-glycerol samples were taken in sterile 10ml Ep tubes, 10. Mu.l of PMA solution was added, mixed by blowing and sucking, incubated in the dark for 5min, and then exposed to intense light from a 650w halogen lamp (placed 25cm from the samples) for 2min, the whole procedure being carried out on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva-glycerol samples. PCR was performed using the DNA as a template and the 6 sets of primers of Table 1, the amplification system is shown in Table 2, and the amplification procedure is shown in Table 3.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, the annealing temperature of the primers of the Proteus, the Fusobacterium, the Porphyromonas, the Treponema and the Tannerella are 58 ℃, and the number of amplification cycles is 35.

Comparative example 1 (fresh saliva collected now)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer tube using a funnel.

Saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. 3ml of the supernatant was pipetted into a sterile 10ml Ep tube, 10. Mu.l of PMA solution was added, mixed by blowing and pipetting, incubated in the dark for 5min, and then exposed to intense light from a 650w halogen lamp (placed 25cm from the sample) for 2min, the whole procedure being carried out on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. PCR was performed using the DNA as a template and the 6 sets of primers of Table 1, the amplification system is shown in Table 2, and the amplification procedure is shown in Table 3.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, the annealing temperature of the primers of the Proteus, the Fusobacterium, the Porphyromonas, the Treponema and the Tannerella are 58 ℃, and the number of amplification cycles is 35.

Agarose gel electrophoresis: according to the size of the DNA sample to be detected, agarose gel with proper concentration is prepared, 6 mu L of mixed sample after PCR amplification is added into a sample application hole, electrophoresis is stopped when an indicator reaches 2/3 of the gel, the gel is soaked in EB solution for about 1min, and then is slowly washed by water, and the electrophoresis result of a UV gel imager is observed, analyzed and recorded.

As shown in figures 1-6, the electrophoresis gel of each group of DNA samples after PCR amplification has little difference in strip brightness, the saliva is preserved for different time according to the saliva preservation method of the invention, the electrophoresis results of the obtained DNA samples are consistent with the current collection of fresh saliva, the saliva DNA samples preserved by the saliva preservation method are all amplified products, and the strip brightness difference is little, which proves that the saliva inoculum preservation method of the invention can well maintain the quantity of surviving bacteria in saliva within a certain time, and the mineral oil can not influence the quality of saliva inoculums. Meanwhile, the 16S universal primer uses all bacteria primers to amplify DNA samples, and all bacteria primers have target bands, so that the saliva inoculum preservation method can well maintain survival of bacteria related to periodontitis in saliva in a certain time.

2. Quantitative PCR detection of surviving bacteria in saliva under different storage conditions

Example 1 (preservation for 0 hours)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer using a funnel, and after completion the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. The freezing tube is placed into a 2.5L round bottom vertical anaerobic culture bag, an outer packing bag of the 2.5L anaerobic gas production bag is torn, the 2.5L anaerobic gas production bag is placed into the freezing tube, two circles of freezing tube are folded along the sealing position of the culture bag, and the freezing tube is fixed by a clamp.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. 3ml of the supernatant was taken in a sterile 10ml Ep tube, 10. Mu.l of PMA solution was added, mixed by blowing and sucking, incubated in the dark for 5min, and then exposed to intense light of a 650w halogen lamp (placed at 25cm from the sample) for 2min, the whole procedure being carried out on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. Real-time fluorescent quantitative PCR amplification was performed using the 6 sets of primers of Table 1 using DNA as a template, the amplification system is shown in Table 4, the amplification procedure is shown in Table 5, and each saliva sample was repeated three times.

Table 4 real-time fluorescent quantitative amplification System

Table 5 real-time fluorescent quantitative amplification procedure

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Wherein the annealing temperature of the universal bacterial primers is 60 ℃, and the annealing temperatures of the primers of the Proteus, the Fusobacterium, the Porphyromonas and the Treponema are 58 ℃.

Example 2 (4 ℃ C. For 4 hours)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer using a funnel, and after completion the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. Placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip; the anaerobic culture bag is put into a refrigerator with the temperature of 4 ℃ for 4 hours.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. 3ml of the supernatant was taken in a sterile 10ml Ep tube, 10. Mu.l of PMA solution was added, mixed by blowing and sucking, incubated in the dark for 5min, and then exposed to intense light of a 650w halogen lamp (placed at 25cm from the sample) for 2min, the whole procedure being carried out on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. Using DNA as a template, it was performed using the 6 sets of primers of Table 1Real-time fluorescent quantitative PCR amplification was performed, the amplification system is shown in Table 4, the amplification procedure is shown in Table 5, and each saliva sample was repeated three times.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, and the annealing temperatures of the primers of the Proteus, the Fusobacterium, the Porphyromonas and the Treponema are 58 ℃.

Example 3 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each volunteer discharged 10ml of unstimulated whole saliva directly into a 15ml sterile freezer using a funnel, and after the end, the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. Placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip; the anaerobic culture bag is put into a refrigerator with the temperature of 4 ℃ for 4 hours.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. The supernatant was dispensed with 50% glycerol, 750. Mu.l of supernatant and 750. Mu.l of glycerol were added to each glycerol tube, and the mixture was air-sucked and mixed, and saliva-glycerol samples were stored in a-80℃refrigerator for 24 hours. After 24 hours, 6ml of salivary glycerin samples were taken in a sterile 10ml Ep tube, 10. Mu.l of PMA solution was added, mixed by blowing and sucking, incubated in the dark for 5min, and then exposed to intense light of a 650w halogen lamp (placed at 25cm from the samples) for 2min, the whole procedure being performed on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva-glycerol samples. Using DNA as a template, it was performed using the 6 sets of primers of Table 1The time-fluorescence quantitative PCR amplification was performed, the amplification system was shown in Table 4, the amplification procedure was shown in Table 5, and each saliva sample was repeated three times.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, and the annealing temperatures of the primers of the Proteus, the Fusobacterium, the Porphyromonas and the Treponema are 58 ℃.

Comparative example 1 (fresh saliva collected now)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer tube using a funnel.

Saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. 3ml of the supernatant was taken in a sterile 10ml Ep tube, 10. Mu.l of PMA solution was added, mixed by blowing and sucking, incubated in the dark for 5min, and then exposed to intense light of a 650w halogen lamp (placed at 25cm from the sample) for 2min, the whole procedure being carried out on ice. By means of

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. Real-time fluorescent quantitative PCR amplification was performed using the 6 sets of primers of Table 1 using DNA as a template, the amplification system is shown in Table 4, the amplification procedure is shown in Table 5, and each saliva sample was repeated three times.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, and the annealing temperatures of the primers of the Proteus, the Fusobacterium, the Porphyromonas and the Treponema are 58 ℃.

Comparative example 2 (saliva was not subjected to PMA treatment after 0 hours of storage)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer using a funnel, and after completion the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. The freezing tube is placed into a 2.5L round bottom vertical anaerobic culture bag, an outer packing bag of the 2.5L anaerobic gas production bag is torn, the 2.5L anaerobic gas production bag is placed into the freezing tube, two circles of freezing tube are folded along the sealing position of the culture bag, and the freezing tube is fixed by a clamp.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. Taking 3ml of supernatant for use

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. Real-time fluorescent quantitative PCR amplification was performed using the 6 sets of primers of Table 1 using DNA as a template, the amplification system is shown in Table 4, the amplification procedure is shown in Table 5, and each saliva sample was repeated three times.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, and the annealing temperatures of the primers of the Proteus, the Fusobacterium, the Porphyromonas and the Treponema are 58 ℃.

Comparative example 3 (saliva samples were not subjected to PMA treatment after 4 hours of storage at 4 ℃)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice.

Each volunteer discharged 10ml of unstimulated whole saliva directly into a 15ml sterile freezer using a funnel, and after the end, the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. Placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip; the anaerobic culture bag is put into a refrigerator with the temperature of 4 ℃ for 4 hours.

Anaerobic removal of sterile cryotubesThe bag was removed and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. Taking 3ml of supernatant for use

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva samples. Real-time fluorescent quantitative PCR amplification was performed using the 6 sets of primers of Table 1 using DNA as a template, the amplification system is shown in Table 4, the amplification procedure is shown in Table 5, and each saliva sample was repeated three times.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, and the annealing temperatures of the primers of the Proteus, the Fusobacterium, the Porphyromonas and the Treponema are 58 ℃.

Comparative example 4 (4 ℃ C. For 4 hours, glycerol-80 ℃ C. For 24 hours, saliva samples were not subjected to PMA treatment)

The periodontitis subjects did not undergo any oral hygiene 24 hours before saliva collection, and did not eat any food or drink for the first 2 hours. The whole saliva collection process was performed on ice. Each subject directly discharged 10ml of unstimulated whole saliva into a 15ml sterile freezer using a funnel, and after completion the lid of the sterile freezer was quickly screwed on. The lid was unscrewed in an ultra clean bench and 1ml of mineral oil was added, taking care not to mix the mineral oil with saliva, mineral oil on top of saliva, and the lid was quickly screwed on. Placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip; the anaerobic culture bag is put into a refrigerator with the temperature of 4 ℃ for 4 hours.

The sterile freezer tube was removed from the anaerobic bag and the upper layer of mineral oil was slowly aspirated with a pipette. The remaining saliva was centrifuged at 2600g for 10min at 4℃to pellet large tissue fragments and eukaryotic cells. The supernatant was dispensed with 50% glycerol, 750. Mu.l of supernatant and 750. Mu.l of glycerol were added to each glycerol tube, and the mixture was air-sucked and mixed, and saliva-glycerol samples were stored at-80℃for 24 hours. After 24 hours, 6ml of saliva-glycerin sample was taken for use

SPIN Kit for Soil genomic DNA extraction kit extracts DNA from saliva-glycerol samples. Real-time fluorescent quantitative PCR amplification was performed using the 6 sets of primers of Table 1 using DNA as a template, the amplification system is shown in Table 4, the amplification procedure is shown in Table 5, and each saliva sample was repeated three times.

Wherein the annealing temperature of the universal bacterial primers is 60 ℃, and the annealing temperatures of the primers of the Proteus, the Fusobacterium, the Porphyromonas and the Treponema are 58 ℃.

As shown in FIG. 7, the Ct values of various kinds of genus of each group are compared, and except for the obvious difference shown in the graph (the obvious difference can show that the saliva inoculant can be well preserved by the method of the invention after being preserved for 24 hours in a refrigerator at-80 ℃ C.), and the bacteria in saliva preserved by the saliva preservation method of the invention are in a survival state and can be used as periodontitis biological film inoculant.

Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims (6)

1. A preservation method of periodontitis biological membrane saliva inoculant, comprising the following steps:

24 hours before saliva collection, periodontitis subjects do not perform any oral hygiene, and do not eat any food or drink in the first 2 hours; the whole saliva collecting process is carried out on ice; each volunteer discharged 10ml of unstimulated whole saliva directly into a 15ml sterile freezer using a funnel, and after the end, the lid of the sterile freezer was quickly screwed on; unscrewing the cover in an ultra clean bench, adding 1ml of mineral oil, taking care that the mineral oil is not mixed with saliva, the mineral oil is arranged on the upper layer of the saliva, and rapidly unscrewing the cover; placing the freezing tube into a 2.5L round bottom vertical anaerobic culture bag, tearing open an outer packing bag of the 2.5L anaerobic gas production bag, placing the outer packing bag into the 2.5L anaerobic gas production bag, folding for two circles along the sealing position of the culture bag, and fixing by using a clip;

after all samples were collected, the anaerobic bags were kept in a refrigerator at 4 ℃ for subsequent saliva inoculum evaluation and inoculation periodontitis biofilm culture.

2. The method for preserving a periodontitis biofilm saliva inoculant according to claim 1, wherein: the funnel and 15ml freezer tube were steam autoclaved at 121 ℃ for 15 minutes before use.

3. The method for preserving a periodontitis biofilm saliva inoculant according to claim 1, wherein: the mineral oil was steam autoclaved at 121 ℃ for 15 minutes before use, followed by sub-packaging in an ultra clean bench.

4. The method for preserving a periodontitis biofilm saliva inoculant according to claim 1, wherein: periodontitis subjects discharged 10ml of saliva into 15ml sterile freezer tubes using a funnel in a sterile room, quickly unscrewed the cap and put on ice.

5. The method for preserving a periodontitis biofilm saliva inoculant according to claim 1, wherein: the sterile freezer is opened at a super clean bench, 1ml of sterilized and packaged mineral oil is slowly added, the mineral oil is not mixed with saliva, and a cover is screwed on the upper layer of the saliva.

6. The method for preserving a periodontitis biofilm saliva inoculant according to claim 1, wherein the sterile freezing tube filled with saliva after oil sealing is placed in a 2.5L round bottom vertical anaerobic culture bag, an outer packing bag of the 2.5L anaerobic gas production bag is torn open, the outer packing bag is placed in the 2.5L anaerobic gas production bag, two circles are folded along the sealing position of the culture bag, and the culture bag is fixed by a clip.

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