CN104306028B - A kind of sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order - Google Patents
A kind of sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order Download PDFInfo
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- 108020004465 16S ribosomal RNA Proteins 0.000 title claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 23
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- 238000001514 detection method Methods 0.000 title claims abstract description 19
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Abstract
The invention discloses a kind of sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order, comprise the following steps: step 1: survey basis forced vital capacity of the first second FEV1；Step 2: suck 200ug albuterol aerosol；Step 3: blowing the nose and 0.9% sterile saline are gargled；Stopping atomization after step 4:3% sterile saline atomization induction 10min, gargle 3 times with 0.9% sterile saline, firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；Step 5: respectively by 4% and 5% sterile saline atomization repeat the above steps, collect deep sputum；Step 6: isolate expectorant bolt the cryopreservation of deep sputum collected by step 4～5.Little, the easy sputum collecting method detected for respiratory tract bacterial 16 S rRNA gene order is polluted by oral cavity it is desirable to provide a kind of.
Description
Technical field
The present invention relates to biomedical sector, a kind of sputum for the detection of respiratory tract bacterial 16 S rRNA gene order is received
Diversity method.
Background technology
Inhaling road microbial population research is a field the newest, and 16S rRNA gene sequencing is to be usually used in mirror at present
Determine the molecular detection technology of bacteria culture, be widely used in the research of environment and clinical samples, respiratory tract microbial population
Research is a field the newest.16S rRNA gene is to encode the corresponding DNA sequence of rRNA on bacterial chromosome,
Being present in the germy chromogene group of institute, the ultimate principle of this technology is to obtain the base of 16S rRNA from micro-biological samples
Because of fragment, obtain 16S rRNA sequence information by clone, order-checking or enzyme action, probe hybridization, then with 16S rRNA data base
In sequence data or other data compare, determine its position in cladogram, thus identify in sample that may be present micro-
Biological species.Accordingly, with respect to traditional antibacterial culturing technology, 16S rRNA sequence analysis can go out the kind of antibacterial by precise Identification
Belong to, and show this kind of antibacterial relative abundance in a part.
How to obtain high-quality lower respiratory tract sputum sample, be the premise of detection respiratory tract 16S rRNA gene sequencing.At present,
The domestic collection method also lacking the effective sputum sample obtaining 16S rRNA gene sequencing.Direct expectoration sputum after gargling,
It is positioned in sterile culture flask, is that current clinic is left and taken sputum, carried out the conventional collection method of antibacterial culturing, has and be easily subject to
The shortcomings such as saliva of buccal cavity and oral cavity bacterium pollute, specimen amount is few and sputum squamous cell is more.In view of induction of sputum 16S rRNA
The importance of gene sequencing technology, and the inherent shortcoming of existing acquisition sputum, it is provided that a kind of the most reliably for 16S
The sputum collecting method of rRNA gene order detection is the most necessary.
Summary of the invention
The present invention provides and is polluted little, the easy sputum collecting side detected for respiratory tract bacterial 16 S rRNA gene order by oral cavity
Method.
The technical scheme that the present invention provides is: a kind of sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order,
Comprise the following steps:
Step 1: survey basis forced vital capacity of the first second FEV1；
Step 2: suck albuterol aerosol；
Step 3: blowing the nose and 0.9% sterile saline are gargled 3 times；
Stopping atomization after step 4:3% sterile saline atomization induction 10min, expectoration first expectorant and saliva abandon, again with 0.9%
Sterile saline is gargled 3 times, and firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
Step: stop atomization after 5:4% sterile saline atomization induction 10min, again gargle 3 times with 0.9% sterile saline,
Firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
Stop atomization after step 6:5% sterile saline atomization induction 10min, again gargle 3 times with 0.9% sterile saline,
Firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
Step 7: isolate expectorant bolt the cryopreservation of deep sputum collected by step 4～6.
This must it is noted that forced vital capacity (forced vital capacity, the FVC) past claim timed vital capacity,
Refer to as possible after maximum air-breathing, the maximum tolerance that can breathe out of exhaling the most as early as possible.It is slightly less than under not free restrictive condition and records
Vital capacity.This index refers to measure the gas ability the most quickly breathed out of vital capacity.Wherein, start to exhale in first second
Expiratory gas volume be one second forced expiratory volume, also known as forced vital capacity of the first second (forced expiratory volume in
One second, FEV1.0), its clinical practice is relatively wide, often represents with FEV1.0/FVC%.
Above-mentioned in the sputum collecting method of respiratory tract bacterial 16 S rRNA gene order detection, described step 2 terminates
After, also include:
FEV1 and forced vital capacity FVC is detected after step 8:15min；
If FEV1/FVC 60%, then carry out step 3；
If 60% > FEV1/FVC 50%, and FEV1 1L, then carry out step 9；
If FEV1/FVC 50%；Or 60%>FEV1/FVC 50%, and FEV1<1L, then terminate deep sputum and collect；
Described step 9 is: blowing the nose and 0.9% sterile saline are gargled 3 times；After 0.9% sterile saline atomization induction 10min
Stopping atomization, again gargle 3 times with 0.9% sterile saline, firmly expectoration deep sputum of holding the breath is collected in sterile petri dish.
Above-mentioned in the sputum collecting method of respiratory tract bacterial 16 S rRNA gene order detection, described step 9 terminates
After, also include:
Step 10: judge whether the weight of the deep sputum collected by step 9 has reached preset value；
The most then terminate deep sputum to collect, isolate expectorant bolt the cryopreservation of deep sputum collected by step 9；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 1, measured in described step 10
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 10 is less than 10%, then carry out step 3；
If the reduction scope of the FEV1 recorded in step 10 is between 10%～20%, then repeat step 9；
If the reduction scope of the FEV1 recorded in step 10 is higher than 20%, then terminates deep sputum and collect.
Above-mentioned in the sputum collecting method of respiratory tract bacterial 16 S rRNA gene order detection, described step 4 terminates
After, also include:
Step 11: judge whether the weight of the deep sputum collected by step 4 has reached preset value；
The most then carry out step 7；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 8, measured in described step 11
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 11 is less than 10%, then carry out step 5；
If the reduction scope of the FEV1 recorded in step 11 is between 10%～20%, then repeat step 4；
If the reduction scope of the FEV1 recorded in step 11 is higher than 20%, then terminates deep sputum and collect.
Above-mentioned in the sputum collecting method of respiratory tract bacterial 16 S rRNA gene order detection, described step 5 terminates
After, also include:
Step 12: after described step 5 terminates, it is judged that whether the weight of the deep sputum collected by step 5 has reached preset value；
The most then carry out step 7；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 1, measured in described step 12
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 12 is less than 10%, then carry out step 6；
If the reduction scope of the FEV1 recorded in step 12 is between 10%～20%, then repeat step 5；
If the reduction scope of the FEV1 recorded in step 12 is higher than 20%, then terminates deep sputum and collect.
Above-mentioned in the sputum collecting method of respiratory tract bacterial 16 S rRNA gene order detection, described step 6 terminates
After, also include:
Step 13: judge whether the weight of the deep sputum collected by step 6 has reached preset value；
The most then carry out step 7；
Collect if it is not, then terminate deep sputum.
Above-mentioned in the sputum collecting method of respiratory tract bacterial 16 S rRNA gene order detection, institute in described step 2
The consumption of the albuterol aerosol stated is 200ug.
Above-mentioned in the sputum collecting method of respiratory tract bacterial 16 S rRNA gene order detection, described preset value is not
Less than 0.1g.
Compared with prior art, step 3,4,5,6 and step 9 all use physiology sterile saline rinse and blowing the nose, fall
The impact on sample of the impurity in low oral cavity, significantly reduces for sputum sample squamous cell and impurity, reduces oral cavity miscellaneous bacteria
The result pollute, tested can fully demonstrate lower respiratory tract flora.And compared to traditional sputum collecting method, this programme
Ladder concentration aerosolized saline is used to improve practical safety.
Accompanying drawing explanation
Fig. 1 is DNA of bacteria electrophoresis result in severe asthma group sputum in embodiment；
Fig. 2 is DNA of bacteria electrophoresis result in mild asthma group sputum in embodiment；
Fig. 3 is DNA of bacteria electrophoresis result in normal healthy controls group sputum in embodiment；
Fig. 4 be severe asthma group in embodiment, mild asthma group and normal healthy controls group sputum in DNA of bacteria sequencing result.
Detailed description of the invention
Below in conjunction with detailed description of the invention, technical scheme is described in further detail, but does not constitute the present invention
Any restriction.
Embodiment 1
1, case is collected
Object of study has 37 examples, wherein severe asthma group 14 example, mild asthma group 14 example, normal healthy controls group 9 example.Serious symptom
The diagnosis of asthma has to comply with the standard on ATS: the symptom repeatedly existed and acute attack, the inhaled steroid of high dose is not
Can alleviate, need frequent oral hormone.Main exclusion standard is: 1.: with the presence of infecting in 4 weeks, orally used anti-in 6 weeks
Raw element；2. there is smoking history；3. merge exist significant pulmonary disease (exist but be not limited to chronic obstructive pulmonary disease,
Tracheaectasy, pulmonary tuberculosis, cystic fibrosis etc.).
2, key instrument and reagent:
Sodium chloride (Guangzhou Chemical Reagent Factory)；Normal saline (Dongguan Pu Ji pharmaceutcal corporation, Ltd)；Sputum extracting genome DNA
Test kit (QIAamp DNA Blood Mini Kit)；Agarose (Biowest company of Spain)；Ultrasound atomizer is (wide
Zhou Yuehua medical apparatus corporation, Ltd)；TGL-16G centrifuge (Anting Scientific Instrument Factory, Shanghai)；Electronic balance (Germany Sartorius
Company)；Accurate pipettor (GILSON company of France)；Lung function instrument (Jaeger company of Germany)；2.0 fluorescence
Meter (American I nvitrogen company)；Electrophresis apparatus (Bio-Rad company of the U.S.).
3, deep sputum is collected and is preserved
Above-mentioned 37 example object of study are carried out deep sputum collection according to the following steps.
Step 1: survey basis forced vital capacity of the first second FEV1；
Step 2: suck albuterol aerosol；
In the present embodiment, after described step 2 terminates, also include:
Detect FEV1 and forced vital capacity FVC after step 8:15min, and judge the ratio of FEV1/FVC, it may appear that following three
The situation of kind:
Situation 1: if FEV1/FVC 60%, then carry out step 3；
Situation 2: if 60% > FEV1/FVC 50%, and FEV1 1L, then carry out step 9；Described step 9 is: blowing the nose and
0.9% sterile saline is gargled 3 times；Atomization is stopped after 0.9% sterile saline atomization induction 10min, again raw with 0.9% sterilizing
Reason saline is gargled 3 times, and firmly expectoration deep sputum of holding the breath is collected in sterile petri dish.
Step 10: judge whether the weight of the deep sputum collected by step 9 has reached preset value；
The most then terminate deep sputum to collect, isolate expectorant bolt the cryopreservation of deep sputum collected by step 9；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 1, measured in described step 10
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 10 is less than 10%, then carry out step 3；
If the reduction scope of the FEV1 recorded in step 10 is between 10%～20%, then repeat step 9；
If the reduction scope of the FEV1 recorded in step 10 is higher than 20%, then terminates deep sputum and collect.
Situation 3: if FEV1/FVC ＜ 50%；Or 60%>FEV1/FVC 50%, and FEV1<1L, then terminate deep sputum and collect；
Above-mentioned 37 example experimentation objects, test respectively according to above-mentioned three kinds of situations, in test process, end do not occur
The situation that only deep sputum is collected.
Step 3: blowing the nose and 0.9% sterile saline are gargled 3 times；
Stopping atomization after step 4:3% sterile saline atomization induction 10min, expectoration first expectorant and saliva abandon, again with 0.9%
Sterile saline is gargled 3 times, and firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
In the present embodiment, after described step 4 terminates, also include:
Step 11: judge whether the weight of the deep sputum collected by step 4 has reached preset value；
The most then carry out step 7；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 8, measured in described step 11
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 11 is less than 10%, then carry out step 5；
If the reduction scope of the FEV1 recorded in step 11 is between 10%～20%, then repeat step 4；
If the reduction scope of the FEV1 recorded in step 11 is higher than 20%, then terminates deep sputum and collect.
Step: stop atomization after 5:4% sterile saline atomization induction 10min, again gargle 3 times with 0.9% sterile saline,
Firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
In the present embodiment, after described step 5 terminates, also include:
Step 12: after described step 5 terminates, it is judged that whether the weight of the deep sputum collected by step 5 has reached preset value；
The most then carry out step 7；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 1, measured in described step 12
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 12 is less than 10%, then carry out step 6；
If the reduction scope of the FEV1 recorded in step 12 is between 10%～20%, then repeat step 5；
If the reduction scope of the FEV1 recorded in step 12 is higher than 20%, then terminates deep sputum and collect.
Stop atomization after step 6:5% sterile saline atomization induction 10min, again gargle 3 times with 0.9% sterile saline,
Firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
In the present embodiment, after described step 6 terminates, also include:
Step 13: judge whether the weight of the deep sputum collected by step 6 has reached preset value；
The most then carry out step 7；
Collect if it is not, then terminate deep sputum.
Step 7: isolate expectorant bolt the cryopreservation of deep sputum collected by step 4～6.
In the present embodiment, the expectorant bolt separating deep sputum described in described step 7 and step 10 the concrete step of cryopreservation
Suddenly it is: by the sticky part in sterilizing tip tweezers picking above-mentioned sterile petri dish sputum in Biohazard Safety Equipment, and to expectorant spigot
Divide and carry out pulling to reduce saliva contamination in sterile petri dish, finally take the sticky sputum of 0.1-0.3g, after putting into sterilizing EP pipe
Weigh, be immediately placed on-80 DEG C of preservations, it is thus achieved that 37 parts of sputum samples.
By aforesaid operations, the sputum weight data of the 37 example object of study collected is as follows:
Table one
4, the extraction of sputum genomic DNA:
QIAamp DNA Mini Kit test kit is used to extract bacterial genomes DNA in sputum as follows.1. sputum sample
In be taken up in order of priority addition TE buffer and the working solution of lysozyme；2. 37 DEG C of water-bath 60min；3. addition protease it is taken up in order of priority
K and AL buffer；4. 95 DEG C of water-bath 5min after 56 DEG C of water-bath 30min；5. stand after adding the anhydrous alcohol that concentration is 99.9%；
6. suspension is transferred to Qiagen separate in pipe；7. it is centrifuged, collects the DNA filtered；8. detergent washing DNA is added；⑨
Dissolve the DNA after washing；10.-20 DEG C of preservations.
Analyze in severe asthma group, mild asthma group and normal healthy controls group bacterial genomes DNA concentration and its degraded journey in sputum
Degree, it is thus achieved that index by be used for evaluating the specimen collected by this kind of induction of sputum in 16S rRNA gene sequencing technology can
By property.
As shown in Figure 1, Figure 2 and Figure 3: severe asthma group, mild asthma group all can become with the induction of sputum in normal healthy controls group
Merit extracts the genomic DNA of antibacterial, and after DNA concentration analysis and sepharose electrophoresis judge, quality is A level, and total amount can
To meet the sample building storehouse needs for 2 times or more than 2 times.
5, genome DNA sample carries out library construction
The genome DNA sample of said extracted carries out library construction: carries out PCR amplification first by merging primer, then uses
Magnetic bead screening fragment, finally, carries out EmPCR amplification and by Roche 454FLX+ platform to whole samples with qualified library
Carry out Manganic pyrophosphate complex initiation.Corresponding analysis of biological information is carried out after machine under pending data.
As shown in Figure 4: result all shows that the DNA of the antibacterial of the sputum of severe asthma group, mild asthma group and normal healthy controls group is equal
Can successfully check order, and draw corresponding biological data.
In the present embodiment, all 37 equal successful acquisition of example object of study have arrived effective deep sputum, by the gradient of the present embodiment
Concentration sterile saline inductive technology can effectively reduce object of study risk, and for severe asthma object of study, the present embodiment is adopted
Proceed by induction by the sterile saline of extremely low concentration (0.9%), further ensure that the safety of object of study.
The 37 parts of samples gathered are complete, affected by saliva little, only exist few squamous cell in microscope, and
Being successfully tested rate high, illustrate to be polluted little by oral cavity bacterium, this programme has extremely strong repeatability.
Above-described be only presently preferred embodiments of the present invention, all made in the range of the spirit and principles in the present invention any amendment,
Equivalent and improvement etc., should be included within the scope of the present invention.
Claims (3)
1. the sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order, it is characterised in that include following
Step:
Step 1: survey basis forced vital capacity of the first second FEV1；
Step 2: suck albuterol aerosol；
After described step 2 terminates, also include:
Step 8: detection FEV1 and forced vital capacity FVC；
If FEV1/FVC 60%, then carry out step 3；
If 60% > FEV1/FVC 50%, and FEV1 1L, then carry out step 9；
If FEV1/FVC ＜ 50%；Or 60%>FEV1/FVC 50%, and FEV1<1L, then terminate deep sputum and collect；
Described step 9 is: blowing the nose and 0.9% sterile saline are gargled 3 times；After 0.9% sterile saline atomization induction 10min
Stopping atomization, again gargle 3 times with 0.9% sterile saline, firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
After described step 9 terminates, also include:
Step 10: judge whether the weight of the deep sputum collected by step 9 has reached preset value；
The most then terminate deep sputum to collect, isolate expectorant bolt the cryopreservation of deep sputum collected by step 9；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 1, measured in described step 10
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 10 is less than 10%, then carry out step 3；
If the reduction scope of the FEV1 recorded in step 10 is between 10%～20%, then repeat step 9；
If the reduction scope of the FEV1 recorded in step 10 is higher than 20%, then terminates deep sputum and collect；
Step 3: blowing the nose and 0.9% sterile saline are gargled 3 times；
Stop atomization after step 4:3% sterile saline atomization induction 10min, again gargle 3 times with 0.9% sterile saline,
Firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
After described step 4 terminates, also include:
Step 11: judge whether the weight of the deep sputum collected by step 4 has reached preset value；
The most then carry out step 7；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 8, measured in described step 11
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 11 is less than 10%, then carry out step 5；
If the reduction scope of the FEV1 recorded in step 11 is between 10%～20%, then repeat step 4；
If the reduction scope of the FEV1 recorded in step 11 is higher than 20%, then terminates deep sputum and collect；
Step: stop atomization after 5:4% sterile saline atomization induction 10min, again gargle 3 times with 0.9% sterile saline,
Firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
After described step 5 terminates, also include:
Step 12: judge whether the weight of the deep sputum collected by step 5 has reached preset value；
The most then carry out step 7；
If it is not, the most again detect FEV1, and judge compared to FEV1 measured in step 1, measured in described step 12
The reduction scope of FEV1；
If the reduction scope of the FEV1 recorded in step 12 is less than 10%, then carry out step 6；
If the reduction scope of the FEV1 recorded in step 12 is between 10%～20%, then repeat step 5；
If the reduction scope of the FEV1 recorded in step 12 is higher than 20%, then terminates deep sputum and collect；
Stop atomization after step 6:5% sterile saline atomization induction 10min, again gargle 3 times with 0.9% sterile saline,
Firmly expectoration deep sputum of holding the breath is collected in sterile petri dish；
After described step 6 terminates, also include:
Step 13: judge whether the weight of the deep sputum collected by step 6 has reached preset value；
The most then carry out step 7；
Collect if it is not, then terminate deep sputum；
Step 7: isolate expectorant bolt the cryopreservation of deep sputum collected by step 4～6.
Sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order the most according to claim 1, it is special
Levying and be, the consumption of the albuterol aerosol described in described step 2 is 200ug.
Sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order the most according to claim 2, it is special
Levying and be, described preset value is no less than 0.1g.
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| CN201410505689.3A CN104306028B (en) | 2014-09-26 | 2014-09-26 | A kind of sputum collecting method for the detection of respiratory tract bacterial 16 S rRNA gene order |
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| CN1191553A (en) * | 1995-07-31 | 1998-08-26 | 查珀尔希尔北卡罗来纳大学 | How to check for lung disease |
| RU2310381C2 (en) * | 2006-01-23 | 2007-11-20 | ГУ Научно-исследовательский институт медицинских проблем Севера СО РАМН | Method for differential diagnostics of bronchial asthma, chronic bronchitis and chronic obstructive pulmonary disease |
| CN201624679U (en) * | 2010-03-19 | 2010-11-10 | 侯文静 | Disposable sputum-taking device |
| CN102192976A (en) * | 2010-03-05 | 2011-09-21 | 复旦大学附属华山医院 | Method for examining and analyzing phlegm, and purpose thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1191553A (en) * | 1995-07-31 | 1998-08-26 | 查珀尔希尔北卡罗来纳大学 | How to check for lung disease |
| RU2310381C2 (en) * | 2006-01-23 | 2007-11-20 | ГУ Научно-исследовательский институт медицинских проблем Севера СО РАМН | Method for differential diagnostics of bronchial asthma, chronic bronchitis and chronic obstructive pulmonary disease |
| CN102192976A (en) * | 2010-03-05 | 2011-09-21 | 复旦大学附属华山医院 | Method for examining and analyzing phlegm, and purpose thereof |
| CN201624679U (en) * | 2010-03-19 | 2010-11-10 | 侯文静 | Disposable sputum-taking device |
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