CN104297309B - The detection electrochemical sensor of pathogenic bacteria, preparation method and applications - Google Patents
The detection electrochemical sensor of pathogenic bacteria, preparation method and applications Download PDFInfo
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Abstract
The present invention provides the detection electrochemical sensor of pathogenic bacteria, preparation method and applications, relates to pathogenic bacteria detection field.The present invention detects the electrochemical sensor of pathogenic bacteria, and including working electrode, described working electrode is the ripple carbon electrode that surface is modified with oligonucleotide probe 2；Described sensor also includes working solution 1 and working solution 2；Described working solution 1 is the nanometer magnetic bead dispersion liquid being marked with anti-pathogenic bacteria antibody；Described working solution 2 is the golden nanometer particle dispersion liquid being marked with anti-pathogenic bacteria antibody and oligonucleotide probe 1；Terminal modified there is methylenum careuleum the 5 ' of described oligonucleotide probe 1, and terminal modified there are amino, the base sequence complementary of described oligonucleotide probe 1 and 2 in the 3 ' of oligonucleotide probe 2.Use inventive sensor detection pathogenic bacteria, convenient and swift, significantly improve detection sensitivity.
Description
Technical field
The present invention relates to pathogenic bacteria detection field, be specifically related to a kind of detect the electrochemical sensor of pathogenic bacteria, system
Preparation Method and application thereof.
Background technology
Pathogenic bacteria (Pathogenic bacteria) refer to cause the microorganism of disease.Pathogenic bacteria include bacterium,
Virus, conveyor screw, rickettsia, Chlamydia, mycoplasma, fungi and actinomyces etc..General described cause
Germ refers to the bacterium in pathogenic microorganism.Pathogenic and its virulence of bacterium, intrusion quantity and portal of entry
Relevant.Although most bacteriums are harmless the most useful, but a large portion can be caused a disease.Condition
Pathogenic bacteria are only caused a disease under given conditions, bacterium can be allowed to enter blood if any wound, or immunity reduces
Time.
Such as: campylobacter jejuni is the bacterium that normally lives away from home of many animals such as ox, sheep, dog and bird.They
Genital tract or enteron aisle have a large amount of bacterium, therefore can be by childbirth or manure contamination food and drinking-water.Campylobacter jejuni
It is a kind of zoonosis pathogen, can cause humans and animals that multiple disease occurs, and be a kind of food source
Property pathogen is it is considered to be cause the main cause that whole world human bacterial property is suffered from diarrhoea.Cardinal symptom for diarrhoea and
Stomachache, generates heat sometimes, occasionally has vomiting and dehydration.Crowd is the most susceptible, and the incidence of disease of less than 5 years old children is the highest,
Summer and autumn is common.Campylobacter jejuni has endotoxin can attack small intestine and colorectal mucosa causes chordapsus, also can draw
Play outbreak of epidemic or the mass food poisoning of diarrhoea.It is generally incubation period 3～5 days, to the pathogenic position of people is
Jejunum, ileum and colon.ETEC (E.coli) is commonly referred to Escherichia coli, is that Escherich is 1885
Year finds, is distributed in nature.Although most Escherichia coli and the mankind have a good cooperation, but still
The Escherichia coli having small part specific type have great virulence, once infect, and will result in serious epidemic situation.
The most representational be exactly code name be the Escherichia coli of O157:H7, it is EHEC (enterohemorrhagic large intestine
Bacillus) a member in family.After human infection EHEC, it may occur that serious spasmodic colic and recurrent exerbation
Hemorrhagic diarrhea, show with heating, vomiting etc. simultaneously, mostly be caused by the toxin of EHEC generation.Some
Severe infections person's toxin causes hemolytic anemia, red blood cell, decrease of platelet with hematogenous spread；Kidney is by ripple
In time it also occur that acute renal failure is the most dead.
The common methods of detection pathogenic bacteria has at present: plating method (Zhang Shuhong, Wu Qingping, Zhang Jumei, Guo Wei
Roc. chromogenic culture medium application [J] in several food-borne pathogens quickly detect. microbiology is circulated a notice of, and 2006,
33 (06): 108-111.), instrumental method (Lv Yanfang, Ma Chunying encourage and build honor. Real-Time Fluorescent Quantitative PCR Technique exists
Food-borne pathogens detection in application [J]. food with fermentation science and technology, 2014,50 (2): 80-84.), immunochromatography
Test strips method (Xia Shiqi, Xu Chaolian, Liu Daofeng, Guo Qi, Wu Songsong, Lai Weihua. colloidal gold immunity chromatography
In joint inspection food 5 kinds of typical salmonella models foundation and optimize [J]. Food Science, 2014,35 (22):
154-158.) etc..But these methods operation complexity, high to detection environment or instrument requirements, the longest, as flat
Plate method is time-consuming and environmental requirement strict, instrumental method not only costliness but also loaded down with trivial details.
Summary of the invention
It is an object of the invention to provide a kind of electrochemical sensor detecting pathogenic bacteria, it is possible to simple, quick,
Sensitive Detection pathogenic bacteria.
It is a further object of the present invention to provide the preparation method of the electrochemical sensor of detection pathogenic bacteria, the method letter
Single, efficiency is high.
Another object of the present invention is to provide the method using described electrochemical sensor detection pathogenic bacteria, the method
Simply, quick, sensitive.
The purpose of the present invention adopts the following technical scheme that realization.
The electrochemical sensor of detection pathogenic bacteria, including working electrode, described working electrode is that surface is modified with widow
The glass-carbon electrode of polynucleotide probe 2；Described sensor also includes working solution 1 and working solution 2；Described working solution 1
For being marked with the nanometer magnetic bead dispersion liquid of anti-pathogenic bacteria antibody；Described working solution 2 is for being marked with anti-pathogenic bacteria antibody
Golden nanometer particle dispersion liquid with oligonucleotide probe 1；The 5 ' of described oligonucleotide probe 1 terminal modified have
Methylenum careuleum, terminal modified there are amino, the base of described oligonucleotide probe 1 and 2 in the 3 ' of oligonucleotide probe 2
Complementary.
In the present invention, the nucleotide sequence (SEQ ID NO:1) of described oligonucleotide probe 1 is
5 '-GTGCACGGTACGCGAATCGG-3 ', the nucleotide sequence (SEQ of described oligonucleotide probe 2
ID NO:2)5’-CCGATTCGCGTACCGTGCAC-3’。
In the present invention, described pathogenic bacteria can be campylobacter jejuni.
The present invention also provides for the preparation method of the electrochemical sensor of described detection pathogenic bacteria, and described surface is modified with
The glass-carbon electrode of oligonucleotide probe 2 is adopted and is prepared with the following method: at glassy carbon electrode surface deposited oxide graphite
Alkene film, modifies oligonucleotide probe 2 on described graphene oxide film.
In the present invention, described surface is modified with the glass-carbon electrode of oligonucleotide probe 2 and adopts and make with the following method
Standby: glass-carbon electrode alumina powder to be polished, second alcohol and water carries out ultrasonic cleaning；By graphene oxide
Suspension dropping glassy carbon electrode surface after ultrasonic cleaning, obtaining surface deposition has the glass of graphene oxide film
Carbon electrode；Surface deposition has the glass-carbon electrode of graphene oxide film at N-hydroxysuccinimide and 1-(3-bis-
Methylaminopropyl)-3-ethyl-carbodiimide hydrochloride solution activates, glass-carbon electrode the most after activation
Surface dropping oligonucleotide probe 2 solution, described oligonucleotide probe 23 ' terminal modified have amino,
Nucleotide sequence (SEQ ID NO:2) is 5 '-CCGATTCGCGTACCGTGCAC-3 '.
In the present invention, described working solution 1 is adopted and is prepared with the following method: nanometer magnetic bead uses 1-(3-diformazan ammonia
Base propyl group)-3-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide activation, be subsequently adding anti-jejunum campylobacter
Bacteria antibody reacts, and obtains being marked with the nanometer magnetic bead dispersion liquid of anti-Antibodies of Campylobacter Jejuni.
In the present invention, described working solution 2 is adopted and is prepared with the following method:
(1) in acetum, add three (2-carboxyethyl) phosphines and 3 ' and terminal modified have sulfydryl, 5 ' terminal modified methylenes
Blue bioprobe, the nucleotide sequence (SEQ ID NO:1) of described bioprobe is
5 '-GTGCACGGTACGCGAATCGG-3 ', react；
(2) in step (1) reaction gained mixture, add golden nanometer particle and react；
(3) in step (2) gained reactant mixture, add anti-Antibodies of Campylobacter Jejuni and react；So
Rear addition deoxyadenosine triphosphate and bovine serum albumin(BSA) react；
(4) step (3) gained reactant mixture is centrifuged, and takes precipitation and is resuspended in phosphate buffer, obtains
It is marked with the golden nanometer particle dispersion liquid of anti-pathogenic bacteria antibody and oligonucleotide probe 1.
The present invention also provides for the method for above-mentioned electrochemical sensor detection pathogenic bacteria, comprises the steps:
(1) working solution 1 is mixed with the solution containing variable concentrations pathogenic bacteria, separate nanometer magnetic bead and suspend
In buffer solution, obtain nanometer magnetic bead dispersion liquid；
(2) in nanometer magnetic bead dispersion liquid, add working solution 2, react；
(3) it is added drop-wise on working electrode react by the mixture that step (2) obtains, cleans, in work
Electrode two ends apply voltage, sensed current signal, obtain the calibration curve of pathogenic bacteria concentration and electric current；
(4) testing sample is detected according to step (1)-(3), the electric current obtained is substituted into standard bent
In line, obtain the concentration of pathogenic bacteria in testing sample.
Compared with prior art, beneficial effects of the present invention is as follows:
The principle of the present invention is as shown in Figure 1.Due to antigen and antibody mediated immunity identification, the present invention is marked with anti-causing a disease
The nanometer magnetic bead of bacteria antibody is equal with the golden nanometer particle being marked with anti-pathogenic bacteria antibody and oligonucleotide probe 1
Can pathogenic bacteria in specific identification sample, define magnetic bead-gold particle compound.Magnetic bead-gold particle is multiple
Oligonucleotide probe 2 complementary pairing that the oligonucleotide probe 1 of compound is modified with working electrode surface, with
Time methylenum careuleum generation redox reaction, thus have electronics Transport And Transformation formed electric current and be detected.The present invention
Employing electrochemistry is as detection means, convenient and swift；Use the nanometer magnetic bead conduct being marked with anti-pathogenic bacteria antibody
The means of enrichment pathogenic bacteria, significantly improve detection sensitivity.The present invention is universal method, it is only necessary to replace
The monoclonal antibody of specific recognition pathogenic bacteria, can realize quick, Site Detection to any pathogenic bacteria, should
Wide by scope, simple application easy to spread.
Accompanying drawing explanation
Fig. 1 shows the principle of detection method.
Fig. 2 is the calibration curve of jejunum campylobacter bacteria concentration and current strength.
Detailed description of the invention
In the present invention, room temperature refers to 20-25 DEG C.
Concentration is the PBS (pH7.4) of 10mM: take 137mmolNaCl, 2.7mmolKCl, 10
mmol Na2HPO4With 2mmol KH2PO4 is dissolved in water, regulates pH to 7.4, is settled to 1L with water.
MES buffer solution: 100mmol 2-(N-morpholine) ethyl sulfonic acid is dissolved in water, regulates pH to 5.9, uses water
It is settled to 1L.
In the present invention, chemical reaction is without open reaction temperature, refers both at room temperature react.
Embodiment 1 detects the electrochemical sensor of campylobacter jejuni
(1) preparation of working solution 1
Working solution 1 is the nanometer magnetic bead dispersion liquid being marked with anti-campylobacter jejuni monoclonal antibody, and nanometer magnetic bead is dense
Degree is 10mmol/L, and nanometer magnetic bead is 1:6 with the mol ratio of anti-campylobacter jejuni monoclonal antibody.
Take 1.08g iron chloride and 0.2g trisodium citrate is dissolved in 20mL ethylene glycol, be subsequently adding 1.2g second
Acid sodium, stirs 30min, puts in 200 DEG C of oil baths and is incubated 10 hours, obtains the nano magnetic that concentration is 10mM
Pearl dispersion liquid.
Take 1-(3-the dimethylamino-propyl)-3-ethyl carbon two of nanometer magnetic bead solution 60 μ L, 0.1mM of 10mM
N-hydroxysuccinimide (NHS) the solution 15 μ L of inferior amine salt hydrochlorate (EDC) solution 15 μ L and 0.01mM,
Mixing, reacts 10-30 minute under room temperature, activates nanometer magnetic bead；Add the anti-jejunum campylobacter of 0.01mg
Bacterium monoclonal antibody is (purchased from abcam, production code member ab31468, anti-campylobacter jejuni antibody titer
1:200, anti-campylobacter jejuni monoclonal antibody affinity costant 1.2 × 1010L/M), mix, anti-under room temperature
Answer 2-4 hour, use magnetic frame Magnetic Isolation nanometer magnetic bead, with the PBS (pH7.4) that concentration is 10mM
Resuspended, obtain the nanometer magnetic bead dispersion liquid of anti-campylobacter jejuni labeling of monoclonal antibody.
(2) preparation of working solution 2
Working solution 2 is the Jenner's grain of rice being marked with anti-campylobacter jejuni monoclonal antibody and oligonucleotide probe 1
Sub-dispersion liquid.In working solution 2, golden nanometer particle concentration is 0.1mg/mL, anti-campylobacter jejuni monoclonal antibody,
Mol ratio between oligonucleotide probe 1 and golden nanometer particle is 1:1:10.Oligonucleotide probe 1
5 ' terminal modified have methylenum careuleum, nucleotides sequence is classified as 5 '-GTGCACGGTACGCGAATCGG-3 '.
The preparation method of golden nanometer particle dispersion liquid: add the chlorauric acid solution of 5mL, 1g/L in conical flask, then use
Aqua sterilisa supplies volume to 50mL, is heated to bumping under stirring, and adding 0.6～1mL concentration is 1% (percent mass
Concentration) sodium citrate solution, continuously stirred, stable to claret to solution colour, obtaining concentration is 0.1mg/mL gold
Nanoparticle dispersion liquid.
Vial adds 2 μ L concentration be the aqueous acetic acid of 500mM, 3 μ L concentration be the three of 1mM
(2-carboxyethyl) phosphine (TCEP) solution and the bioprobe 1 that 20 μ L concentration are 1nM, hybrid reaction 1 hour.
It is subsequently adding 200 μ L and concentrates the golden nanometer particle dispersion liquid (pH 8.0) of 10 times, hybrid reaction 1 hour.Then
Adding the anti-campylobacter jejuni monoclonal antibody (source is ibid) that 100 μ L concentration are 0.1mg/mL, mixing is anti-
Answer 1 hour.Add deoxyadenosine triphosphate (dATP) solution that 20 μ L concentration are 100 μMs and 20 μ L,
10% (w/v) bovine serum albumin solution, hybrid reaction half an hour.By reactant mixture, 9.4g from
It is centrifuged 7 minutes under mental and physical efforts, takes precipitation and be resuspended in 500 μ LPBS buffer solution (10mM, pH7.4), obtain
It is marked with the golden nanometer particle dispersion liquid of anti-campylobacter jejuni monoclonal antibody and oligonucleotide probe 1, places
In 4 DEG C of environment, storage.
Bioprobe 1: at 3 ' the terminal modified sulfydryls of nucleotide fragments 5 '-GTGCACGGTACGCGAATCGG-3 ',
5 ' terminal modified methylenum careuleum (MB), structural formula is MB-5 '-GTGCACGGTACGCGAATCGG-3 '-SH, by upper
Hai Shenggong biotech firm synthesizes.
(3) preparation of working electrode
Working electrode is the glass-carbon electrode that surface is modified with oligonucleotide probe 2.
Glass-carbon electrode particle diameter is the alumina powder polishing of 0.05 micron, then at absolute ethyl alcohol and ultra-pure water
Ultrasonic 2 minutes of middle difference.
Weigh 25mg graphene oxide and join in the PBS solution that 50mL concentration is 0.1mol/L, then
Within ultrasonic 30 minutes, form it into the uniform brown oxidation Graphene suspension that concentration is 0.5mg/mL.
Drip 20 μ L above-mentioned graphene oxide suspension in glassy carbon electrode surface, be dried, in glassy carbon electrode surface
Deposited oxide graphene film.
NaOH that concentration is 1.5mol/L is molten to have the glass-carbon electrode of graphene oxide film to be placed in surface deposition
In liquid, react 5 minutes under 1.5V voltage.Take out glass-carbon electrode, drip 10 μ L at electrode surface and contain
100mmol/L N-hydroxysuccinimide (NHS) and 400mmol/L 1-(3-dimethylamino-propyl)-3-ethyl
2-(N-morpholine) ethyl sulfonic acid (MES) buffer solution (0.1mol/L, pH5.9) of carbodiimide hydrochloride (EDC),
Carry out priming reaction 1 hour, then use MES buffer solution for cleaning.
Electrode surface after activation drips the oligonucleotide probe 2 that 10 μ L concentration are 25 μMs, reacts 1
Hour, clean with PBS (10mM, pH7.4), it is thus achieved that working electrode.
3 ' ends of oligonucleotide probe 2 have decorations amino, and nucleotides sequence is classified as
5 '-CCGATTCGCGTACCGTGCAC-3 ', structural formula is 5 '-CCGATTCGCGTACCGTGCAC-3 '-NH2 +,
Synthesized by Shanghai Sheng Gong biotech firm.
(4) Ag/AgCl is reference electrode, and platinum electrode is auxiliary electrode.
Embodiment 2 detects the campylobacter jejuni in actual sample
1. Criterion curve
Use the electrochemical sensor detection campylobacter jejuni detecting campylobacter jejuni in embodiment 1.Detection process
The most at room temperature.
(1) take 20 μ L working solutions 1 join 1mL be added with variable concentrations campylobacter jejuni (ATCC33560,
Purchased from Central Plains, Beijing company) milk soln in, be placed on after mixing on rotary mixer, at 25 revs/min
Under the conditions of, react 10 minutes.Use magnetic frame Magnetic Isolation nanometer magnetic bead-campylobacter jejuni compound, remove
Supernatant, adds PBS (pH 7.4) solution that 1mL concentration is 10mM, and is placed on rotary mixer cleaning 3
Minute, repeated washing 3 times afterwards, it is eventually adding 100 μ L PBS (10mM, pH 7.4) resuspended,
Obtain nanometer magnetic bead-campylobacter jejuni compound dispersion liquid.
(2) respectively take 20 μ L immunomagnetic beadses-campylobacter jejuni compound dispersion liquid and working solution 2 mixes, 20
React 30 minutes under the conditions of rev/min, it is thus achieved that magnetic bead-gold particle complex solution.
(3) drip 10 μ L magnetic beads-gold particle complex solution at working electrode surface, react 1 hour, use
PBS (10mM, pH 7.4) cleans.
(4) Ag/AgCl is reference electrode, and platinum electrode is auxiliary electrode, uses Differential Pulse Voltammetry,
Electrode two ends apply 0.0 volt to-0.4 volt voltage, the electric current that detection working electrode produces.Obtain campylobacter jejuni dense
Degree and the calibration curve (Fig. 2) of current strength, it can be seen that the detection range of detection method is 5-400
cfu/mL。
2. concrete sample detection
Concrete sample uses the present embodiment title 1 method detect, the signal of telecommunication (electric current) value will be recorded and bring in mark song.
SEQUENCE LISTING
<110>
Propagation and Food Test Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau
<120>
The detection electrochemical sensor of pathogenic bacteria, preparation method and applications
<130>
20141029
<160>
2
<170>
PatentIn version 3.3
<210>
1
<211>
20
<212>
DNA
<213>
artificial
<220>
<223>
The nucleotide sequence of oligonucleotide probe 1
<400>
1
gtgcacggta
cgcgaatcgg
20
<210>
2
<211>
20
<212>
DNA
<213>
artificial
<220>
<223>
The nucleotide sequence of oligonucleotide probe 2
<400>
2
ccgattcgcg
taccgtgcac
20
Claims (8)
1. detect the electrochemical sensor of pathogenic bacteria, including working electrode, it is characterised in that described working electrode
The glass-carbon electrode of oligonucleotide probe 2 it is modified with for surface；Described sensor also includes working solution 1 and working solution
2；Described working solution 1 is the nanometer magnetic bead dispersion liquid being marked with anti-pathogenic bacteria antibody；Described working solution 2 is mark
There is the golden nanometer particle dispersion liquid of anti-pathogenic bacteria antibody and oligonucleotide probe 1；Described oligonucleotide probe 1
5 ' terminal modified have methylenum careuleum, terminal modified there are amino, described oligonucleotide in the 3 ' of oligonucleotide probe 2
The base sequence complementary of probe 1 and 2.
Detect the electrochemical sensor of pathogenic bacteria the most according to claim 1, it is characterised in that described oligomerization core
The nucleotides sequence of thuja acid probe 1 is classified as 5 '-GTGCACGGTACGCGAATCGG-3 ', described oligonucleotides
The nucleotide sequence 5 '-CCGATTCGCGTACCGTGCAC-3 ' of acid probe 2.
The electrochemical sensor of detection pathogenic bacteria the most according to claim 1 or claim 2, it is characterised in that described cause
Germ is campylobacter jejuni.
4. detect the preparation method of the electrochemical sensor of pathogenic bacteria described in claim 1, it is characterised in that described
Surface is modified with the glass-carbon electrode of oligonucleotide probe 2 and adopts and prepare with the following method: sink in glassy carbon electrode surface
Long-pending graphene oxide film, modifies oligonucleotide probe 2 on described graphene oxide film.
Detect the preparation method of the electrochemical sensor of pathogenic bacteria the most according to claim 4, it is characterised in that
Described surface is modified with the glass-carbon electrode of oligonucleotide probe 2 and adopts and prepare with the following method: used by glass-carbon electrode
Alumina powder polishes, and carries out ultrasonic cleaning in second alcohol and water；Graphene oxide suspension is dripped ultrasonic
Glassy carbon electrode surface after cleaning, obtaining surface deposition has the glass-carbon electrode of graphene oxide film；Surface deposits
There is the glass-carbon electrode of graphene oxide film at N-hydroxysuccinimide and 1-(3-dimethylamino-propyl)-3-ethyl
Carbodiimide hydrochloride solution activates, glassy carbon electrode surface dropping oligonucleotide the most after activation
Probe 2 solution, the 3 ' of described oligonucleotide probe 2 terminal modified have amino, nucleotides sequence to be classified as
5’-CCGATTCGCGTACCGTGCAC-3’。
6. according to claim 4 or 5, detect the preparation method of the electrochemical sensor of pathogenic bacteria, its feature
It is that described working solution 1 is adopted to prepare with the following method: nanometer magnetic bead uses 1-(3-dimethylamino-propyl)-3-ethyl
Carbodiimide hydrochloride and the activation of N-hydroxysuccinimide, be subsequently adding anti-Antibodies of Campylobacter Jejuni and carry out instead
Should, obtain being marked with the nanometer magnetic bead dispersion liquid of anti-Antibodies of Campylobacter Jejuni.
Detect the preparation method of the electrochemical sensor of pathogenic bacteria the most according to claim 6, it is characterised in that
Described working solution 2 is adopted and is prepared with the following method:
(1) in acetum, add three (2-carboxyethyl) phosphines and 3 ' and terminal modified have sulfydryl, 5 ' terminal modified methylenes
Blue bioprobe, the nucleotides sequence of described bioprobe is classified as
5 '-GTGCACGGTACGCGAATCGG-3 ', react；
(2) in step (1) reaction gained mixture, add golden nanometer particle and react；
(3) in step (2) gained reactant mixture, add anti-Antibodies of Campylobacter Jejuni and react；So
Rear addition deoxyadenosine triphosphate and bovine serum albumin(BSA) react；
(4) step (3) gained reactant mixture is centrifuged, and takes precipitation and is resuspended in phosphate buffer, obtains
It is marked with the golden nanometer particle dispersion liquid of anti-pathogenic bacteria antibody and oligonucleotide probe 1.
8. the method using one of claim 1-3 described electrochemical sensor detection pathogenic bacteria, comprises the steps:
(1) working solution 1 is mixed with the solution containing variable concentrations pathogenic bacteria, separate nanometer magnetic bead and suspend
In buffer solution, obtain nanometer magnetic bead dispersion liquid；
(2) in nanometer magnetic bead dispersion liquid, add working solution 2, react；
(3) it is added drop-wise on working electrode react by the mixture that step (2) obtains, cleans, in work
Electrode two ends apply voltage, sensed current signal, obtain the calibration curve of pathogenic bacteria concentration and electric current；
(4) testing sample is detected according to step (1)-(3), the electric current obtained is substituted into standard bent
In line, obtain the concentration of pathogenic bacteria in testing sample.
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