Method for quantitatively detecting viable and non-culturable bacteria in respiratory tract of host and application

Abstract

The invention discloses a method for quantitatively detecting viable and non-culturable bacteria in a respiratory tract of a host and application. According to the method, a respiratory tract bacterium sample is treated by utilizing a designed specific primer in combination with a PMA dye and a real-time fluorescent quantitative PCR technology, and specific bacteria in a living and non-culturable state in the respiratory tract of a host can be accurately and quantitatively detected by the method. Compared with a flow cytometry and a method for detecting living non-culturable bacteria by ATP (adenosine triphosphate), the method disclosed by the invention has the advantages that a certain target bacterium in a complex bacterium sample which is extracted in a host body and is low in concentration and interfered by other microbial components can be accurately quantified, and the experiment accuracy and repeatability are high; and the defect that the traditional detection method cannot distinguish the detection of the viable and non-culturable bacteria of the single bacteria can be overcome. The method provided by the invention can realize rapid and accurate detection of a plurality of viable and non-culturable bacteria in a clinical sample, and can be applied to human health risk assessment.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular, relates to a method and application for quantitatively detecting living unculturable bacteria in the respiratory tract of a host. Background Art

[0002] Many studies have pointed out that air pollution is the main environmental factor causing the global disease burden, causing millions of premature deaths each year. Lower respiratory tract infection is the world's deadliest infectious disease and the fourth leading cause of death. Since bacteria that enter the respiratory tract will enter a dormant state due to antibiotic attack or host defense, the drug resistance of dormant bacteria will be greatly increased, leading to the persistence and recurrence of respiratory infections. In harsh environments or nutrient deficiencies, bacteria reduce energy consumption and prolong survival time by entering a dormant state. Bacteria use dormancy to escape environmental threats. Viable but non-culture (VBNC) bacteria, as a state of existence of dormant bacteria that can escape host attack and elimination, have a great impact on human health, thus attracting special attention.

[0003] Although studies have elucidated the mechanism of bacterial variation in vitro, there is little information about phenotypic variation in vivo. However, due to the complexity of the in vivo environment, the collected samples are not only of low concentration, but also usually contain complex microbial individuals other than the target bacteria, such as host cell fragments and other pathogens. However, the current detection methods for live uncultivable bacteria are mainly: ① Detecting bacterial vitality indicators through ATP kits, but the detection process will be interfered by non-microbial sources, resulting in false positive results. ② Determining the bacterial state by flow cytometry, but based on flow cytometry, it is impossible to distinguish the number of a certain type of bacteria in complex bacterial samples. In addition, the concentration of samples required for flow cytometry is high, but the concentration of samples in organisms is generally low. Therefore, when the concentration of samples in organisms is very low, it is impossible to use flow cytometry to detect live uncultivable bacteria. Therefore, there is an urgent need for a method that can accurately and quantitatively detect the state and quantity of a certain bacteria at a lower concentration in vivo. Summary of the invention

[0004] The object of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art, and to provide a method for detecting bacteria that have entered a dormant state in an intact animal host when exposed to bacteria, and specifically, to provide a method for quantitatively detecting live, unculturable bacteria in the respiratory tract of a host.

[0005] The second object of the present invention is to provide application of the method.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention first provides a method for quantitatively detecting live unculturable bacteria in the respiratory tract of a host, wherein the bacteria are any one or more of Pseudomonas aeruginosa, Escherichia coli or Staphylococcus aureus; the method comprises the following steps:

[0008] S1. Collect bacterial samples from the host's respiratory tract;

[0009] S2. PMA-qPCR is used to quantitatively detect the number of live bacteria in the bacterial sample; wherein the primers specific for Pseudomonas aeruginosa are selected from any pair of nucleotide sequences such as SEQ ID No. 1-2, 3-4, 5-6, the primers specific for Escherichia coli are selected from any pair of nucleotide sequences such as SEQ ID No. 7-8, 9-10, and the primers specific for Staphylococcus aureus are selected from any pair of nucleotide sequences such as SEQ ID No. 11-12, 13-14;

[0010] S3. Subtract the number of culturable bacteria from the number of live bacteria obtained in step S2 to obtain the number of live, unculturable bacteria in the bacterial sample.

[0011] In view of the problem of low bacterial concentration in the host, the present invention designs and screens specific primers for target bacteria, obtains specific primers for Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, and combines PMA dye and qPCR to effectively quantitatively detect the number of bacteria entering the VBNC state under the conditions of interference from complex microbial flora in the host respiratory tract and low concentration of target bacteria. The detection concentration range of the method is 1.0×10 3 ~3.0×10 9 CFU / mL. The method of the present invention can be applied to the quantitative detection of live uncultivable bacteria in the host, which is conducive to a deep understanding of the interaction mechanism between the host and bacteria and effectively avoids the potential risks brought by it.

[0012] Furthermore, the host in step S1 includes but is not limited to mammalian bodies such as rats or humans.

[0013] Furthermore, the respiratory tract in step S1 is any one or more parts of the nasal passage, throat, trachea and lungs.

[0014] Furthermore, the bacterial sample in step S1 is collected using a sterile phosphate buffer solution.

[0015] Preferably, the concentration of the sterile phosphate buffer solution is 0.1 mol / L.

[0016] Preferably, when the host to be detected is a rat, the step S1 is to bleed the rat to death, and collect bacterial samples from the nasal passage, throat, trachea, and lungs; wherein, the nasal passage sampling method is to open the cheeks of the rat on both sides, cut off the lower jaw and remove it, expose the visible posterior nasal passage opening on the upper jaw of the rat, use an lavage needle to take 1.0 to 30.0 mL of a 0.1 mol / L sterile phosphate buffer solution to lavage from the posterior nasal passage forward three times, collect the lavage fluid, and obtain the bacterial sample to be tested in the nasal passage; the throat sampling method is to use a disposable flocked swab to gently wipe the side wall and tonsils of the pharynx to avoid touching the tongue, teeth or other parts of the oral cavity to reduce contamination, and the sampling is repeated 3 times, and the first collection is The flocked swab is immersed in 1.0-50.0mL of 0.1mol / L sterile phosphate buffer solution and shaken to obtain the pharyngeal swab bacterial sample to be tested; the tracheal sampling method is to cut the trachea open, wipe the inner wall of the trachea with a disposable flocked swab, repeat three times, and soak the sampled swab in 1.0-50.0mL of 0.1mol / L sterile phosphate buffer solution, shaken to obtain the tracheal bacterial sample to be tested; the lung sampling method is to make a T-shaped opening along the rat's throat to expose the trachea, take 1.0-100.0mL of 0.1mol / L sterile phosphate buffer solution for 3 times to perform alveolar lavage on the lungs, and obtain the lung bacterial sample to be tested.

[0017] Preferably, when the host to be detected is a human body, the step S1 is to collect bacterial samples from the nasal passage, pharynx, trachea and lungs of the human body; wherein, the nasal passage sampling method is to use a disposable flocked swab to rotate and wipe the nasal cavity to collect bacterial samples, the sampling is repeated 3 times, and the collected disposable flocked swab is immersed in 1.0-50.0mL of a sterile phosphate buffer solution with a concentration of 0.1mol / L, and shaken to obtain the bacterial sample to be tested in the nasal passage; the throat sampling method is to use a disposable flocked swab to gently wipe the side wall and tonsils of the pharynx to avoid touching the tongue, teeth or other parts of the oral cavity to reduce contamination, and the sampling is repeated 3 times, and the collected disposable flocked swab is immersed in 1.0-50.0mL of a sterile phosphate buffer solution with a concentration of 0.1mol / L, and shaken to obtain the bacterial sample to be tested in the nasal passage; the throat sampling method is to use a disposable flocked swab to gently wipe the side wall and tonsils of the pharynx to avoid touching the tongue, teeth or other parts of the oral cavity to reduce contamination, and the sampling is repeated 3 times. The pharyngeal swab is added to 0mL of 0.1mol / L sterile phosphate buffer solution and shaken to obtain the bacterial sample to be tested from the pharyngeal swab; the sampling method for the trachea and lungs is sputum sampling (suitable for people who can sputum spontaneously) or bronchoscope sampling. If sputum sampling is adopted, the sampling method is to rinse the mouth with clean water several times, take a deep breath and hold the breath for 3 seconds, forcefully cough out deep sputum (not less than 1.0mL), spit it into a sterile sputum box, and liquefy the sputum to obtain the bacterial sample to be tested from the trachea or lungs; if bronchoscopic sampling is used, the subject needs to be locally anesthetized, a bronchoscope is inserted through the nasal cavity or oral cavity, an appropriate amount of sterile saline is injected, aspirated back, and the lavage fluid is collected to obtain the bacterial sample to be tested from the trachea or lungs.

[0018] Furthermore, the concentration range of the quantitative detection of the number of viable bacteria in step S2 is 1.0×10 3 ~3.0×10 9 CFU / mL.

[0019] Furthermore, the step of PMA-qPCR quantitative detection in step S2 includes performing qPCR detection using bacterial DNA solutions of known different concentrations to construct a standard curve of Ct value-DNA concentration; incubating the bacterial sample with PMA dye in the dark, extracting the DNA in the bacterial sample after photolysis, performing qPCR detection, and comparing the obtained Ct value with the standard curve to obtain the number of live bacteria in the bacterial sample.

[0020] Preferably, the method for constructing the standard curve is to prepare OD 600nm =1.0, extract bacterial DNA, use different concentration gradients of bacterial DNA to perform real-time fluorescence quantitative PCR amplification, and draw a standard curve of bacterial Ct value with respect to DNA concentration.

[0021] Preferably, the light-proof incubation time is 1.0 to 30.0 min.

[0022] Preferably, the light-proof incubation time is 15.0 to 30.0 min.

[0023] Preferably, the photolysis time is 1.0 to 30.0 min.

[0024] Preferably, the photolysis time is 15.0 to 30.0 min.

[0025] Preferably, the photolysis is performed using a PL-PMA photolyzer.

[0026] Furthermore, the number of culturable bacteria in step S3 is obtained by counting by a spread plate method.

[0027] The present invention provides the use of any of the above methods in clinical detection of live unculturable bacteria for non-disease diagnosis purposes.

[0028] The present invention provides application of any of the above methods in human health risk assessment for purposes other than disease diagnosis.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a method for quantitatively detecting live uncultivable bacteria in the host's respiratory tract. The method comprises incubating PMA dye mixed with a bacterial sample, and using a designed specific primer, combined with real-time fluorescence quantitative PCR technology to process the respiratory bacterial sample. This method can accurately and quantitatively detect specific (Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus) bacteria in a live uncultivable state in the host's respiratory tract. Compared with the method of detecting live uncultivable bacteria by flow cytometry and ATP, the advantage of this method is that it can accurately quantify a certain target bacterium in a complex bacterial sample with low concentration extracted from the host and interference from other microbial components. The experimental accuracy and repeatability are high, which can make up for the defect that the traditional detection method cannot distinguish the detection of live uncultivable bacteria of a single bacterium. The method of the present invention can realize rapid and accurate detection of multiple live uncultivable bacteria in clinical samples, and is applied in human health risk assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the melting curve of the primers specific to Pseudomonas aeruginosa.

[0032] Figure 2 This is the melting curve of the E. coli specific primer.

[0033] Figure 3 This is the melting curve of the Staphylococcus aureus-specific primer.

[0034] Figure 4 Flow chart for the quantitative detection of viable, unculturable bacteria in the rat respiratory tract.

[0035] Figure 5 This is a standard curve of PMA-qPCR performed using Pseudomonas aeruginosa in Example 2.

[0036] Figure 6 This is the dissolution curve of bacterial samples from the nasal cavity, pharynx, trachea, and lungs amplified using specific primers for Pseudomonas aeruginosa in Example 2.

[0037] Figure 7 The growth conditions of the bacterial samples isolated from the host's respiratory nasal cavity, pharynx, trachea, and lungs in Example 2 on cetyl trimethylammonium bromide agar medium.

[0038] Figure 8 This is the recovery rate of nasal lavage fluid and alveolar lavage fluid samples collected from SD rats in Example 3.

[0039] Fig. 9 The figure shows the number of live uncultivable Pseudomonas aeruginosa in samples from various parts of the respiratory tract collected from SD rats in Example 3. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] The photolyzer model used in the embodiment of the present invention is a PL-PMA photolyzer, and the DNA extraction kit model is a Biospin bacterial genomic DNA extraction kit.

[0043] Example 1

[0044] Since the respiratory tract contains a variety of microorganisms and the concentration of extracted bacteria is low, this method designs specific primers for different bacteria and screens them, so that when the concentration of sample bacteria is low, the interference of other bacteria can be eliminated and the number of bacteria in the VBNC state of one of the bacteria can be quantified. The specific method is as follows:

[0045] 1. Primer design: The present invention designs different specific primers for Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus (Table 1) and uses them for PMA-qPCR detection:

[0046] Table 1 Design of bacteria-specific primers

[0047]

[0048]

[0049] 1. Standard curve drawing: Culture Pseudomonas aeruginosa / Escherichia coli / Staphylococcus aureus overnight and adjust the OD with sterile PBS 600nm =1.0. Take 1.0 mL of the above bacterial solution for DNA extraction, and adjust it with sterile water to DNA solutions of different concentrations for real-time fluorescence quantitative PCR detection. Use the Ct values ​​obtained by DNA amplification to draw a standard curve.

[0050] 2. PMA binding: Take 1.0 mL of bacterial samples from various parts of the respiratory tract of SD rats at the end of exposure, add propidium azide bromide dye (PMA) and incubate in the dark for 15.0 minutes, then put it in a photolyzer for photolysis for 10.0 minutes.

[0051] 3.qPCR reaction:

[0052] ①Prepare premix

[0053] The premix solution is prepared according to the ratio of 10.0μL qPCR Mix, 0.4μL forward primer, 0.4μL reverse primer, and 7.2μL Nuclease-Free Water per well. Taking into account the loss during pipetting, the actual volume of the premix solution should be prepared according to the volume of several more sample wells than the theoretically required volume.

[0054] ② In the eight-tube strip, add 18.0 μL of premix and 2.0 μL of DNA template to each well, and set up 3 replicate wells for each sample. After adding the sample, vortex to remove bubbles and put it into the qPCR instrument for subsequent experiments.

[0055] ③Set up the program

[0056] The qPCR program was set as a two-step method, including pre-denaturation, denaturation, and annealing & extension, where the pre-denaturation temperature was 95°C for 10 min, the denaturation temperature was 95°C for 10 s, and the annealing & extension temperature was 60°C for 30 s. 40 cycles were repeated from denaturation to annealing and extension.

[0057] The reaction system of qPCR is shown in Table 2, and the specific steps of qPCR are shown in Table 3:

[0058] Table 2 qPCR reaction system

[0059]

[0060] Table 3 qPCR reaction steps

[0061]

[0062] The melting curves of primer sequences for Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus are shown in Figure 2. Figures 1 to 3 As shown, a single melting curve peak represents good specificity, so primer pairs 1 to 3 were selected as specific primers for detecting Pseudomonas aeruginosa, primer pairs 6 to 7 were specific primers for Escherichia coli, and primer pairs 9 to 10 were specific primers for Staphylococcus aureus.

[0063] Example 2

[0064] The flow chart of the method for quantitatively detecting viable non-culturable (VBNC) bacteria in the respiratory tract of rats using the PMA-qPCR method is as follows Figure 4 shown.

[0065] 1. After the bacterial exposure, the SD rats were anesthetized and killed. The side walls and tonsils of the pharynx were gently wiped with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity. The disposable flocked swab after sampling was soaked in 10.0 mL of 0.1 mol / L sterile phosphate buffer solution and placed in a shaker for full shaking to obtain the pharyngeal swab bacterial sample to be tested.

[0066] 2. Open the throat of the rat killed in step 1 in a T-shape to expose the trachea, take 30.0 mL of 0.1 mol / L sterile phosphate buffer and lavage with a lavage needle three times to obtain the alveolar lavage bacterial sample to be tested.

[0067] 3. The trachea of ​​the rat killed in step 1 was cut open, and the inside of the trachea was sampled with a disposable flocked swab. The sampled swab was immersed in 5.0 mL of a sterile phosphate buffer solution with a concentration of 0.1 mol / L, and placed in a shaker for shaking to obtain a tracheal bacterial sample to be tested.

[0068] 4. Open the cheeks of the rats killed in step 1, cut off the lower jaw and remove it, expose the upper jaw of the rat and make the posterior nasal opening visible. Use an lavage needle to take 15 mL of 0.1 mol / L sterile phosphate buffer solution and lavage from the posterior nasal meatus to the front three times. Collect the lavage fluid in a centrifuge tube to obtain the nasal lavage bacterial sample to be tested.

[0069] 5. Cultivate Pseudomonas aeruginosa overnight, wash with sterile phosphate buffer solution and adjust the OD 600nm = about 1.0, take 1.0mL of the bacterial solution for DNA extraction, and adjust the DNA concentration with sterile water to prepare DNA solutions with different concentration gradients, use the DNA solutions with different concentrations for real-time fluorescence quantitative PCR detection, and use the obtained Ct values ​​to draw a standard curve of Ct value with respect to DNA concentration.

[0070] 6. Take 1.0 mL of the bacterial sample to be tested in step 1, step 2, step 3, and step 4 respectively into a sterile centrifuge tube and wrap the centrifuge tube with tin foil. Under light-proof conditions, take 1.0 μL of PMA dye and add it to each centrifuge tube. Place it on a rocker and incubate it in the dark for 15.0 min. Then remove the tin foil wrapping the centrifuge tube and place it in the PL-PMA photolysis instrument for illumination for 15.0 min before taking it out.

[0071] 7. Use a DNA extraction kit to extract the bacterial sample treated in step 6. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0072] 8. Use the specific primers of Pseudomonas aeruginosa (SEQ ID No. 1-2), mix, and NF-Water to prepare the premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 7 to each well. Set the appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of the samples in each part, and compare it with the standard curve in step 5 ( Figure 5 ) to calculate the number of viable target bacteria in the sample Figure 6 The melting curve of the sample amplified with the specific primers of Pseudomonas aeruginosa. In addition, according to the equation of the standard curve, Y = -3.334 × X + 43.98 (R 2 =0.9921), it can be deduced that the concentration range of live bacteria of Pseudomonas aeruginosa detectable by the present invention is 1.0×10 3 ~3.0×10 9 CFU / mL.

[0073] 9. Take 100.0 μL of the bacterial suspension obtained in step 1, step 2, step 3, and step 4, and spread it on hexadecyl trimethylammonium bromide agar medium to count the number of culturable Pseudomonas aeruginosa per milliliter ( Figure 7 ). Subtract the number of culturable bacteria from the number of viable bacteria obtained in step 8 to obtain the number of viable, unculturable Pseudomonas aeruginosa per milliliter.

[0074] Example 3

[0075] 1. After the bacterial exposure, the SD rats were anesthetized and killed. The side walls and tonsils of the pharynx were gently wiped with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity. The disposable flocked swab after sampling was soaked in 20.0 mL of 0.1 mol / L sterile phosphate buffer solution and placed in a shaker for full shaking to obtain the pharyngeal swab bacterial sample to be tested.

[0076] 2. Open the throat of the rat killed in step 1 in a T-shape to expose the trachea, take 60.0 mL of 0.1 mol / L sterile phosphate buffer and lavage with a lavage needle three times to obtain the alveolar lavage bacterial sample to be tested.

[0077] 3. The trachea of ​​the rat killed in step 1 was cut open, and the inside of the trachea was sampled with a disposable flocked swab. The sampled swab was immersed in 10.0 mL of a 0.1 mol / L sterile phosphate buffer solution and placed in a shaker to obtain a tracheal bacterial sample to be tested.

[0078] 4. Open the cheeks of the rats killed in step 1, cut off the lower jaw and remove it, expose the upper jaw of the rat and make the posterior nasal cavity opening visible. Use an lavage needle to take 30.0 mL of 0.1 mol / L sterile phosphate buffer solution to lavage from the posterior nasal cavity forward three times, and collect the lavage fluid in a centrifuge tube to obtain the nasal lavage bacterial sample to be tested.

[0079] 5. Cultivate Pseudomonas aeruginosa overnight, wash with sterile phosphate buffer solution and adjust the OD 600nm = about 1.0, take 1.0mL of the bacterial solution for DNA extraction, and adjust the DNA concentration with sterile water to prepare DNA solutions with different concentration gradients, use the DNA solutions with different concentrations for real-time fluorescence quantitative PCR detection, and use the obtained Ct values ​​to draw a standard curve of Ct value with respect to DNA concentration.

[0080] 6. Take 1.0 mL of the bacterial sample to be tested in step 1, step 2, step 3, and step 4 respectively into a sterile centrifuge tube and wrap the centrifuge tube with tin foil. Under light-proof conditions, take 1.0 μL of PMA dye and add it to each centrifuge tube. Place it on a rocker and incubate it in the dark for 30.0 min. Then remove the tin foil wrapping the centrifuge tube and place it in a PL-PMA photolysis instrument for 30.0 min of illumination before taking it out.

[0081] 7. Use a DNA extraction kit to extract the bacterial sample treated in step 6. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0082] 8. Use specific primers for Pseudomonas aeruginosa (SEQ ID No.3-4), mix, and NF-Water to prepare a premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 7 to each well. Set an appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of each sample. Compare it with the standard curve in step 5 to calculate the number of live bacteria of the target bacteria in the sample.

[0083] 9. Take 100.0 μL of the bacterial suspension obtained in step 1, step 2, step 3, and step 4, and spread it on the hexadecyl trimethylammonium bromide agar medium to count the number of culturable Pseudomonas aeruginosa per milliliter. Subtract the number of culturable bacteria from the number of live bacteria obtained in step 8 to obtain the number of live unculturable Pseudomonas aeruginosa per milliliter.

[0084] Figure 8 To collect the recovery rate of nasal lavage fluid and alveolar lavage fluid samples from SD rats, Fig. 9 The graph shows the number of live, unculturable Pseudomonas aeruginosa in samples from various parts of the respiratory tract collected from SD rats.

[0085] Example 4

[0086] 1. After the bacterial exposure, the SD rats were anesthetized and killed. The side walls and tonsils of the pharynx were gently wiped with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity. The disposable flocked swab after sampling was soaked in 10.0 mL of 0.1 mol / L sterile phosphate buffer solution and placed in a shaker for full shaking to obtain the pharyngeal swab bacterial sample to be tested.

[0087] 2. Open the throat of the rat killed in step 1 in a T-shape to expose the trachea, take 30.0 mL of 0.1 mol / L sterile phosphate buffer and lavage with a lavage needle three times to obtain the alveolar lavage bacterial sample to be tested.

[0088] 3. The trachea of ​​the rat killed in step 1 was cut open, and the inside of the trachea was sampled with a disposable flocked swab. The sampled swab was immersed in 5.0 mL of a 0.1 mol / L sterile phosphate buffer solution and placed in a shaker to obtain a tracheal bacterial sample to be tested.

[0089] 4. Open the cheeks of the rats killed in step 1, cut off the lower jaw and remove it, expose the upper jaw of the rat and make the posterior nasal opening visible. Use an lavage needle to take 15.0 mL of 0.1 mol / L sterile phosphate buffer solution and lavage from the posterior nasal meatus to the front three times. Collect the lavage fluid in a centrifuge tube to obtain the nasal lavage bacterial sample to be tested.

[0090] 5. Grow E. coli overnight, wash with sterile phosphate buffer solution and adjust OD 600nm = 1.0, 1.0 mL of the bacterial solution was taken for DNA extraction, and the DNA concentration was adjusted with sterile water to prepare DNA solutions with different concentration gradients. The DNA solutions with different concentrations were used for real-time fluorescence quantitative PCR detection, and the obtained Ct values ​​were used to draw a standard curve of Ct value with respect to DNA concentration. According to the standard curve, it was deduced that the concentration range of live Escherichia coli detectable by the present invention was 1.0×10 3 ~3.0×10 9 CFU / mL.

[0091] 6. Take 1.0 mL of the bacterial sample to be tested in step 1, step 2, step 3, and step 4 respectively into a sterile centrifuge tube and wrap the centrifuge tube with tin foil. Under light-proof conditions, take 1.0 μL of PMA dye and add it to each centrifuge tube. Place it on a rocker and incubate it in the dark for 15.0 min. Then remove the tin foil wrapping the centrifuge tube and place it in the PL-PMA photolysis instrument for illumination for 15.0 min before taking it out.

[0092] 7. Use a DNA extraction kit to extract the bacterial sample treated in step 6. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0093] 8. Use Escherichia coli specific primers (SEQ ID No.7-8), mix, and NF-Water to prepare a premix, add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 7 to each well, set an appropriate real-time fluorescence quantitative PCR program, amplify the DNA of the target live bacteria to obtain the Ct value of each sample, compare it with the standard curve in step 5, and calculate the number of live bacteria of the target bacteria in the sample.

[0094] 9. Take 100 μL of the bacterial suspension obtained in step 1, step 2, step 3, and step 4, spread it on LB agar medium and count the number of culturable E. coli per milliliter. Subtract the number of culturable bacteria from the number of live bacteria obtained in step 8 to get the number of live, unculturable E. coli per milliliter.

[0095] Example 5

[0096] 1. After the bacterial exposure, the SD rats were anesthetized and killed. The side walls and tonsils of the pharynx were gently wiped with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity. The disposable flocked swab after sampling was soaked in 10.0 mL of 0.1 mol / L sterile phosphate buffer solution and placed in a shaker for full shaking to obtain the pharyngeal swab bacterial sample to be tested.

[0097] 2. Open the throat of the rat killed in step 1 in a T-shape to expose the trachea, take 20.0 mL of 0.1 mol / L sterile phosphate buffer and lavage with a lavage needle three times to obtain the alveolar lavage bacterial sample to be tested.

[0098] 3. The trachea of ​​the rat killed in step 1 was cut open, and the inside of the trachea was sampled with a disposable flocked swab. The sampled swab was immersed in 10.0 mL of a 0.1 mol / L sterile phosphate buffer solution and placed in a shaker to obtain a tracheal bacterial sample to be tested.

[0099] 4. Open the cheeks of the rats killed in step 1, cut off the lower jaw and remove it, expose the upper jaw of the rat and make the posterior nasal opening visible. Use an lavage needle to take 10.0 mL of 0.1 mol / L sterile phosphate buffer solution and lavage from the posterior nasal meatus to the front three times. Collect the lavage fluid in a centrifuge tube to obtain the nasal lavage bacterial sample to be tested.

[0100] 5. Grow E. coli overnight, wash with sterile phosphate buffer solution and adjust OD 600nm= about 1.0, take 1.0mL of the bacterial solution for DNA extraction, and adjust the DNA concentration with sterile water to prepare DNA solutions with different concentration gradients, use the DNA solutions with different concentrations for real-time fluorescence quantitative PCR detection, and use the obtained Ct values ​​to draw a standard curve of Ct value with respect to DNA concentration.

[0101] 6. Take 2 mL of the bacterial samples to be tested in step 1, step 2, step 3, and step 4 respectively into sterile centrifuge tubes and wrap the centrifuge tubes with tin foil. Under light-proof conditions, take 2.0 μL of PMA dye and add it to each centrifuge tube. Place them on a rocker and incubate them in the dark for 30.0 min. Then remove the tin foil wrapping the centrifuge tubes and place them in a PL-PMA photolyzer for 30.0 min of illumination before taking them out.

[0102] 7. Use a DNA extraction kit to extract the bacterial sample treated in step 6. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0103] 8. Use Escherichia coli specific primers (SEQ ID No. 9-10), mix, and NF-Water to prepare a premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 7 to each well. Set an appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of each sample. Compare it with the standard curve in step 5 to calculate the number of live bacteria of the target bacteria in the sample.

[0104] 9. Take 100.0 μL of the bacterial suspension obtained in step 1, step 2, step 3, and step 4, spread it on LB agar medium and count the number of culturable E. coli per milliliter. Subtract the number of culturable bacteria from the number of live bacteria obtained in step 8 to get the number of live unculturable E. coli per milliliter.

[0105] Example 6

[0106] 1. After the bacterial exposure, the SD rats were anesthetized and killed. The side walls and tonsils of the pharynx were gently wiped with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity. The disposable flocked swab after sampling was soaked in 10.0 mL of 0.1 mol / L sterile phosphate buffer solution and placed in a shaker for full shaking to obtain the pharyngeal swab bacterial sample to be tested.

[0107] 2. Open the throat of the rat killed in step 1 in a T-shape to expose the trachea, take 30.0 mL of 0.1 mol / L sterile phosphate buffer and lavage with a lavage needle three times to obtain the alveolar lavage bacterial sample to be tested.

[0108] 3. The trachea of ​​the rat killed in step 1 was cut open, and the inside of the trachea was sampled with a disposable flocked swab. The sampled swab was immersed in 5.0 mL of a 0.1 mol / L sterile phosphate buffer solution and placed in a shaker to obtain a tracheal bacterial sample to be tested.

[0109] 4. Open the cheeks of the rats killed in step 1, cut off the lower jaw and remove it, expose the upper jaw of the rat and make the posterior nasal opening visible. Use an lavage needle to take 15.0 mL of 0.1 mol / L sterile phosphate buffer solution and lavage from the posterior nasal meatus to the front three times. Collect the lavage fluid in a centrifuge tube to obtain the nasal lavage bacterial sample to be tested.

[0110] 5. Cultivate Staphylococcus aureus overnight, wash with sterile phosphate buffer solution and adjust the OD 600nm = 1.0, 1.0 mL of the bacterial solution was taken for DNA extraction, and the DNA concentration was adjusted with sterile water to prepare DNA solutions with different concentration gradients. The DNA solutions with different concentrations were used for real-time fluorescence quantitative PCR detection, and the obtained Ct values ​​were used to draw a standard curve of Ct value with respect to DNA concentration. According to the standard curve, it was deduced that the concentration range of live Staphylococcus aureus detectable by the present invention was 1.0×10 3 ~3.0×10 9 CFU / mL.

[0111] 6. Take 2.0 mL of the bacterial samples to be tested in step 1, step 2, step 3, and step 4 respectively into sterile centrifuge tubes and wrap the centrifuge tubes with tin foil. Under light-proof conditions, take 2.0 μL of PMA dye and add it to each centrifuge tube. Place them on a rocker and incubate them in the dark for 15.0 min. Then remove the tin foil wrapping the centrifuge tubes and place them in a PL-PMA photolyzer for illumination for 15.0 min before taking them out.

[0112] 7. Use a DNA extraction kit to extract the bacterial sample treated in step 6. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0113] 8. Use specific primers for Staphylococcus aureus (SEQ ID No. 11-12), mix, and NF-Water to prepare a premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 7 to each well. Set an appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of each sample. Compare it with the standard curve in step 5 to calculate the number of live bacteria of the target bacteria in the sample.

[0114] 9. Take 100 μL of the bacterial suspension obtained in step 1, step 2, step 3, and step 4, spread it on LB agar medium and count the number of culturable Staphylococcus aureus per milliliter. Subtract the number of culturable bacteria from the number of live bacteria obtained in step 8 to get the number of live unculturable Staphylococcus aureus per milliliter.

[0115] Example 7

[0116] 1. After the bacterial exposure, the SD rats were anesthetized and killed. The side walls and tonsils of the pharynx were gently wiped with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity. The disposable flocked swab after sampling was soaked in 25.0 mL of 0.1 mol / L sterile phosphate buffer solution and placed in a shaker for full shaking to obtain the pharyngeal swab bacterial sample to be tested.

[0117] 2. Open the throat of the rat killed in step 1 in a T-shape to expose the trachea, take 50.0 mL of 0.1 mol / L sterile phosphate buffer and lavage with a lavage needle three times to obtain the alveolar lavage bacterial sample to be tested.

[0118] 3. The trachea of ​​the rat killed in step 1 was cut open, and the inside of the trachea was sampled with a disposable flocked swab. The sampled swab was immersed in 15.0 mL of a sterile phosphate buffer solution with a concentration of 0.1 mol / L, and placed in a shaker for shaking to obtain a tracheal bacterial sample to be tested.

[0119] 4. Open the cheeks of the rats killed in step 1, cut off the lower jaw and remove it, expose the upper jaw of the rat and make the posterior nasal cavity opening visible. Use an lavage needle to take 25.0 mL of 0.1 mol / L sterile phosphate buffer solution and lavage from the posterior nasal cavity to the front three times. Collect the lavage fluid in a centrifuge tube to obtain the nasal lavage bacterial sample to be tested.

[0120] 5. Cultivate Staphylococcus aureus overnight, wash with sterile phosphate buffer solution and adjust the OD 600nm = about 1.0, take 1.0mL of the bacterial solution for DNA extraction, and adjust the DNA concentration with sterile water to prepare DNA solutions with different concentration gradients, use the DNA solutions with different concentrations for real-time fluorescence quantitative PCR detection, and use the obtained Ct values ​​to draw a standard curve of Ct value with respect to DNA concentration.

[0121] 6. Take 2.0 mL of the bacterial samples to be tested in step 1, step 2, step 3, and step 4 respectively into sterile centrifuge tubes and wrap the centrifuge tubes with tin foil. Under light-proof conditions, take 2.0 μL of PMA dye and add it to each centrifuge tube. Place them on a rocker and incubate them in the dark for 15.0 min. Then remove the tin foil wrapping the centrifuge tubes and place them in a PL-PMA photolyzer for illumination for 15.0 min before taking them out.

[0122] 7. Use a DNA extraction kit to extract the bacterial sample treated in step 6. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0123] 8. Use specific primers for Staphylococcus aureus (SEQ ID No. 13-14), mix, and NF-Water to prepare a premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 7 to each well. Set an appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of each sample. Compare it with the standard curve in step 5 to calculate the number of live bacteria of the target bacteria in the sample.

[0124] 9. Take 100.0 μL of the bacterial suspension obtained in step 1, step 2, step 3, and step 4, spread it on LB agar medium and count the number of culturable Staphylococcus aureus per milliliter. Subtract the number of culturable bacteria from the number of live bacteria obtained in step 8 to get the number of live unculturable Staphylococcus aureus per milliliter.

[0125] Example 8

[0126] 1. Collect bacterial samples from the human nasal passages. The sampling method is to use a disposable flocked swab to rotate and wipe the nasal cavity to collect bacterial samples. The sampling is repeated 3 times. The collected disposable flocked swab is soaked in 1.0-50.0mL of a sterile phosphate buffer solution with a concentration of 0.1mol / L and shaken to obtain the bacterial sample to be tested in the nasal passages.

[0127] 2. Collect bacterial samples from the human throat. The sampling method is to gently wipe the side walls and tonsils of the pharynx with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity to reduce contamination. The sampling is repeated 3 times. The collected disposable flocked swab is immersed in 1.0-50.0 mL of a sterile phosphate buffer solution with a concentration of 0.1 mol / L and shaken to obtain the pharyngeal swab bacterial sample to be tested.

[0128] 3. Collect bacterial samples from the human trachea and lungs. The sampling method is sputum sampling (suitable for people who can cough up sputum on their own) or bronchoscope sampling. If sputum sampling is used, the sampling method is to rinse the mouth with clean water several times, take a deep breath and hold the breath for 3 seconds, cough out deep sputum (not less than 1.0mL) with force, spit it into a sterile sputum box, and liquefy the sputum to obtain the trachea or lung bacterial sample to be tested; if bronchoscopic sampling is used, the subject needs to be locally anesthetized, and a bronchoscope is inserted through the nasal cavity or oral cavity. 30.0-40.0mL of sterile saline is injected each time, and it is aspirated back and repeated 3 times. The lavage fluid is collected to obtain the trachea or lung bacterial sample to be tested.

[0129] 4. Cultivate Pseudomonas aeruginosa overnight, wash with sterile phosphate buffer solution and adjust the OD 600nm = about 1.0, take 1.0mL of the bacterial solution for DNA extraction, and adjust the DNA concentration with sterile water to prepare DNA solutions with different concentration gradients, use the DNA solutions with different concentrations for real-time fluorescence quantitative PCR detection, and use the obtained Ct values ​​to draw a standard curve of Ct value with respect to DNA concentration.

[0130] 5. Take 1.0 mL of the bacterial sample to be tested in step 1, step 2, and step 3 respectively into a sterile centrifuge tube and wrap the centrifuge tube with tin foil. Under light-proof conditions, take 1.0 μL of PMA dye and add it to each centrifuge tube. Place it on a rocker and incubate it in the dark for 30.0 min. Then remove the tin foil wrapped around the centrifuge tube and place it in the PL-PMA photolysis instrument for 30.0 min of illumination before taking it out.

[0131] 6. Use a DNA extraction kit to extract the bacterial sample treated in step 5. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0132] 7. Use specific primers for Pseudomonas aeruginosa (SEQ ID No. 1-2), mix, and NF-Water to prepare a premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 6 to each well. Set an appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of each sample. Compare it with the standard curve in step 4 to calculate the number of live bacteria of the target bacteria in the sample.

[0133] 8. Take 100.0 μL of the bacterial suspension obtained in step 1, step 2, and step 3, and spread it on the cetyl trimethylammonium bromide agar medium to count the number of culturable Pseudomonas aeruginosa per milliliter. Subtract the number of culturable bacteria from the number of live bacteria obtained in step 7 to obtain the number of live unculturable Pseudomonas aeruginosa per milliliter.

[0134] Application Example 1

[0135] 1. Select a group of clinical patients' sputum samples and liquefy the sputum to facilitate subsequent DNA extraction operations.

[0136] 2. Cultivate Pseudomonas aeruginosa overnight, wash with sterile phosphate buffer solution and adjust the OD 600nm = about 1.0, take 1.0mL of the bacterial solution for DNA extraction, and adjust the DNA concentration with sterile water to prepare DNA solutions with different concentration gradients, use the DNA solutions with different concentrations for real-time fluorescence quantitative PCR detection, and use the obtained Ct values ​​to draw a standard curve of Ct value with respect to DNA concentration.

[0137] 3. Take 2.0 mL of the liquefied sputum sample in step 1 and put it into a sterile centrifuge tube. Wrap the centrifuge tube with tin foil. Under light-proof conditions, take 2.0 μL of PMA dye and add it to the centrifuge tube. Place it on a rocker and incubate it in the dark for 15.0 minutes. Then remove the tin foil wrapped around the centrifuge tube and place it in the PL-PMA photolyzer for 15.0 minutes of illumination before taking it out.

[0138] 4. Use a DNA extraction kit to extract the DNA from the sputum bacterial sample in step 1. The extracted DNA is immediately used for downstream experiments or stored at -20.0°C.

[0139] 5. Use specific primers for Pseudomonas aeruginosa (SEQ ID No. 1-2), mix, and NF-Water to prepare a premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 4 to each well. Set an appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of each sample. Compare it with the standard curve in step 2 to calculate the number of live bacteria of the target bacteria in the sample.

[0140] 6. Take 100.0 μL of the sputum sample in step 1, spread it on the hexadecyl trimethylammonium bromide agar medium and count the number of culturable Pseudomonas aeruginosa per milliliter of sputum sample. The number of live Pseudomonas aeruginosa in the non-culturable state per milliliter of sputum sample can be obtained by subtracting the number of culturable bacteria from the number of live bacteria obtained in step 5. According to the above method, the number of live non-culturable bacteria of Escherichia coli / Staphylococcus aureus can also be obtained respectively using the corresponding bacteria-specific primers and the corresponding culturable bacteria counts.

[0141] 7. Combine the test results with the clinical treatment response to evaluate the guiding value of quantitative detection of VBNC bacteria for clinical treatment.

[0142] Application Example 2

[0143] 1. Expose SD rats to a solution containing 10 8 CFU / m 3 aeruginosa in the environment and control the exposure time.

[0144] 2. After the bacterial exposure, the SD rats were anesthetized and killed, and the side walls and tonsils of the pharynx were gently wiped with a disposable flocked swab to avoid touching the tongue, teeth or other parts of the oral cavity. The disposable flocked swab after sampling was soaked in 10.0 mL of 0.1 mol / L sterile phosphate buffer solution and placed in a shaker for full shaking to obtain the pharyngeal swab bacterial sample to be tested.

[0145] 3. Open the throat of the rat killed in step 2 in a T-shape to expose the trachea, take 30.0 mL of 0.1 mol / L sterile phosphate buffer and lavage with a lavage needle three times to obtain the alveolar lavage bacterial sample to be tested.

[0146] 4. The trachea of ​​the rat killed in step 2 was cut open, and the inside of the trachea was sampled with a disposable flocked swab. The sampled swab was immersed in 5.0 mL of a sterile phosphate buffer solution with a concentration of 0.1 mol / L, and placed in a shaker for shaking to obtain a tracheal bacterial sample to be tested.

[0147] 5. Open the cheeks of the rats killed in step 2, cut off the lower jaw and remove it, expose the upper jaw of the rat and make the posterior nasal opening visible. Use an lavage needle to take 10.0 mL of 0.1 mol / L sterile phosphate buffer solution and lavage from the posterior nasal meatus to the front three times. Collect the lavage fluid in a centrifuge tube to obtain the nasal lavage bacterial sample to be tested.

[0148] 6. Cultivate Pseudomonas aeruginosa overnight, wash with sterile phosphate buffer solution and adjust the OD 600nm = about 1.0, take 1.0mL of the bacterial solution for DNA extraction, and adjust the DNA concentration with sterile water to prepare DNA solutions with different concentration gradients, use the DNA solutions with different concentrations for real-time fluorescence quantitative PCR detection, and use the obtained Ct values ​​to draw a standard curve of Ct value with respect to DNA concentration.

[0149] 7. Take 2.0 mL of the bacterial samples to be tested in step 2, step 3, step 4, and step 5 respectively into sterile centrifuge tubes and wrap the centrifuge tubes with tin foil. Under light-proof conditions, take 2.0 μL of PMA dye and add it to each centrifuge tube. Place them on a rocker and incubate them in the dark for 15.0 min. Then remove the tin foil wrapping the centrifuge tubes and place them in a PL-PMA photolysis instrument for illumination for 15.0 min before taking them out.

[0150] 8. Use a DNA extraction kit to extract the bacterial sample treated in step 7. The extracted DNA can be used immediately for downstream experiments or stored at -20.0°C.

[0151] 9. Use specific primers of Pseudomonas aeruginosa (SEQ ID NO.1-2), mix, and NF-Water to prepare a premix. Add 18.0 μL of the premix and 2.0 μL of the DNA sample in step 8 to each well. Set an appropriate real-time fluorescence quantitative PCR program to amplify the DNA of the target live bacteria to obtain the Ct value of the samples in each part, compare it with the standard curve in step 6, and calculate the number of live bacteria of the target bacteria in the sample.

[0152] 10. Take 100.0 μL of the bacterial suspension obtained in step 2, step 3, step 4, and step 5, spread it on the hexadecyl trimethylammonium bromide agar medium, and count the number of culturable Pseudomonas aeruginosa per milliliter. The number of live Pseudomonas aeruginosa in an unculturable state per milliliter can be obtained by subtracting the number of culturable bacteria from the number of live bacteria obtained in step 9. According to the above method, the number of live unculturable bacteria of Escherichia coli / Staphylococcus aureus can also be obtained respectively using the corresponding bacteria-specific primers and the corresponding culturable bacteria counts.

[0153] 11. Regularly monitor the weight changes, behavioral responses and physiological indicators of exposed animals. After the experiment, dissect the animals to check for pathological changes in the lungs and other related organs, such as alveolar wall thickening, red blood cell accumulation in the alveolar cavity, etc. Combine pathological results with physiological monitoring data to assess the potential risks of Pseudomonas aeruginosa exposure to human health.

[0154] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A method for quantitatively detecting live, unculturable bacteria in the respiratory tract of a host for non-disease diagnosis purposes, characterized in that: The bacteria are any one or more of Pseudomonas aeruginosa, Escherichia coli or Staphylococcus aureus; the method comprises the following steps: S1. Collect bacterial samples from the host's respiratory tract; S2. PMA-qPCR is used to quantitatively detect the number of live bacteria in the bacterial sample; wherein the primers specific for Pseudomonas aeruginosa are selected from any pair of nucleotide sequences such as SEQ ID No. 1-2, 3-4, 5-6, the primers specific for Escherichia coli are selected from any pair of nucleotide sequences such as SEQ ID No. 7-8, 9-10, and the primers specific for Staphylococcus aureus are selected from any pair of nucleotide sequences such as SEQ ID No. 11-12, 13-14; S3. Subtract the number of culturable bacteria in the bacterial sample from the number of live bacteria obtained in step S2 to obtain the number of live, unculturable bacteria in the bacterial sample.

2. The method according to claim 1, characterized in that: The host in step S1 is selected from rats or humans.

3. The method according to claim 1, characterized in that: The respiratory tract in step S1 is any one or more parts of the nasal passage, throat, trachea and lungs.

4. The method according to claim 1, characterized in that: The bacterial sample in step S1 is collected using a sterile phosphate buffer solution.

5. The method according to claim 1, characterized in that: The concentration range of the quantitative detection of the number of viable bacteria in step S2 is 1.0×10 3 ~3.0×10 9 CFU / mL.

6. The method according to claim 1, characterized in that: The step of PMA-qPCR quantitative detection in step S2 includes performing qPCR detection using bacterial DNA solutions of known different concentrations to construct a standard curve of Ct value-DNA concentration; incubating the bacterial sample with PMA dye in the dark, extracting DNA from the bacterial sample after photolysis, performing qPCR detection, and comparing the obtained Ct value with the standard curve to obtain the number of live bacteria in the bacterial sample.

7. The method according to claim 6, characterized in that: The light-proof incubation time is 1 to 30 minutes.

8. The method according to claim 6, characterized in that: The photolysis time is 1 to 30 minutes.

9. The method according to claim 1, characterized in that: The number of culturable bacteria in step S3 is obtained by counting by the spread plate method.

10. Use of the method according to any one of claims 1 to 9 in human health risk assessment for purposes other than disease diagnosis.

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