CN107523607A - It is a kind of to be analyzed with Protocols in Molecular Biology and quantify probiotics and the method for pathogen thalline quantity - Google Patents
It is a kind of to be analyzed with Protocols in Molecular Biology and quantify probiotics and the method for pathogen thalline quantity Download PDFInfo
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Abstract
It is a kind of to be analyzed with Protocols in Molecular Biology and quantify probiotics and the method for pathogen thalline quantity, comprise the steps of：Step 1:Take an inspection product to be dissolved in sterilized water, with high speed centrifugation, by thalline under, sterilized water dilution is added, as bacterium sample to be measured；Step 2:Bacterium sample to be measured is with real-time quantitative polymerase chain reaction collocation object bacteria specific primers group；The wherein object bacteria specific primers group is the conservative gene with object bacteria, including 16S rDNA, 23S rDNA, and for 16S 23S rDNA interior transcriptional domain or housekeeping gene sequence fragment as amplification target, the wherein object bacteria is probiotics or pathogen；Selectivity crest is whether there is with the melting curve of amplified fragments, determines whether the bacterium sample to be measured contains non-targeted strain；Step 3:Bring the inspection product Ct values of acquisition the regression equation of standard curve made from standard items into, the thalline quantity of bacterium sample to be measured can be learnt.
Description
Technical field
The present invention is to be analyzed on one kind with Real-time round pcrs and quantify probiotics and pathogen thalline quantity
Method, and the thalline quantity contained with the derivative commodity of this technical Analysis.
Background technology
Polymerase chain reaction (English：Polymerase chain reaction, abbreviation：PCR) mainly DNA is used
Three steps such as denaturation, bonding and extension circulation, continuously expand target DNA fragments, amplify the DNA of denier, it is fixed in real time
It is that fluorescence probe or dyestuff are utilized during PCR to measure PCR (Real-time PCR), then reaches via optical system and detect in real time
Survey the effect of (Real-time).Each time after PCR amplification cycles, the quantity of its reaction of formation product (fluorescent material) is recorded
Get off, after question response is fully completed, mapping is drawn with amplification cycles number (cycle number) and generation product amount, can be obtained
To a response curve figure, the generation situation of each amplification cycles (cycle) product in complete presentation PCR reactions, so
Referred to as real-time quantitative PCR.By analyzing PCR cycle and generating the relativeness between product amount the two factors, to carry out
The interpretation of experimental result.
The frequency that Real-time PCR are used in recent years is gradually enhanced, and advantage can be significantly comprising (1) experimental periods
Shorten, (2) can monitor experimental result in real time on computers, and selectivity, susceptibility and the degree of accuracy that (3) is identified are more traditional
PCR modes are high, and (4) detection process does not have to carry out agar gel electrophoresis, the material except that can save time and agar glue
Outside taking, it can also avoid doing the caused pollution of agar gel electrophoresis experiment and operating error more.In comparison, normal PCR
Qualitative analysis is simply possible to use in, the degree of " sxemiquantitative (semi-quantitative) " is reached to multipotency.
In recent years, there are many documents or patented technology, the quantity of probiotics included with PCR analyses or Quantifying Bacteria,
It is described as follows.
The A of People's Republic of China (PRC) application for a patent for invention publication No. CN 101717828, a kind of patent name " acidified milk system
The method that kinds of lactobacillus is quickly examined in product ", the method first extract the DNA of lactic acid bacteria in fermented dairy product, and by given zone
Domain dna carries out amplified reaction, and this PCR primer is analyzed with gel electrophoresis.Mark is compared made of electrophoresis result and reference strain
Floating screed band is compared, and determines the species contained in sample.The method is except needing manual operation to carry out standard band ratio
It is right, also can only be qualitative by lactic acid bacteria culturers, but can not quantify.
The A of People's Republic of China (PRC) application for a patent for invention publication No. CN 103525914, " one kind counts plant to patent name
The method and its primer and kit of living preparation of lactobacillus number ", the method need first that sample is (single with plating medium culture single bacterium colony
The bacterium colony that individual bacterium is formed), and the DNA for extracting viable bacteria bacterium colony enters performing PCR reaction.Product utilization gel electrophoresis is identified.Quantitative scoring
Number is may bacterium number using colony counting method and PCR reaction ratio conversions.The method is except needing first to turn out single bacterium with flat board
Fall, and can only be qualitative by lactic acid bacteria culturers, but can not quantify.
The B of People's Republic of China (PRC) invention patent mandate notification number CN 102994644, a kind of patent name " plant breast bar
Bacterium quantitative detecting method and its test kit and application ", the method need to extract the total serum IgE of testing sample, bright with ultraviolet spectrometry
Degree meter measure light absorption value, calculates copy number and is diluted, and by RNA reverse transcriptions into cDNA, then real-time quantitative PCR is carried out, utilize
Have specialized plant lactobacillus primer pair, fluorescent PCR amplification is carried out by masterplate of gained cDNA.The method is only detected in sample and deposited
Lactobacillus plantarum quantity living.
Document " research of molecular beacon real-time PCR methodology quick detection Bifidobacterium " (microbiology circular 2007), is utilized
Real-time PCR are analyzed and are quantified the number of active bifid bacteria, the strain of the probiotics of exposure, design of primers, all with the present invention
Difference, and active bacteria number can only be analyzed.
Defend the inspection of the double fork Bacillus acidi lacticis of Food microbe testing method-lactic acid bacteria-lactic acid of good fortune portion bulletin, this inspection in Taiwan
The bacterium identification of lactic acid lactobacillus bifidus (Bifidobacterium lactis) in proved recipe method scope of application food, but the method
Without standard measure lactic acid bacteria bacterium number.
World patent cooperation treaty (PCT) WO2010151124, patent name Selection of a nutritional
Composition capable of promoting health (screening for promoting the composition of health-nutrition), disclose and utilize
PCR carries out probiotics strain idenfication, and this invention is qualitative merely with PCR progress lactic acid bacterias, and without standard measure lactic acid bacteria bacterium number.
Document " Development of a Quantitative PCR for Detection of Lactobacillus
plantarum Starters During Wine Malolactic
Fermentation.J.Microbiol.Biotechnol. (2011), 21 (12), 1280-1286 ", disclosed in
In the malic acid of Grauburgunder wine, lactic fermentation process, develop quantitative real-time PCR method and be used to count
Lactobacillus plantarum IWBT B188, qRT-PCR have bacterial strain selectivity, are according to plastid DNA sequence or seat
The plantaricin gene orders (L.plantarum-specific) for falling within chromosome are the primer sets of target.The method its
Minimum detecting limit is 1 × 102-1×103CFU/mL, the counting Lactobacillus plantarum bacterium available for selectivity
Kind.But the primer amplified region that the method uses only plantaricin genes, and viable count can only be measured.
Document " Quantitative PCR for the specific quantification of Lactococcus
lactis and Lactobacillus paracasei and its interest for Lactobacillus lactis
in cheese samples.Food Microbiol.2013Dec；36(2):286-95 ", disclose a kind of real-time quantitative PCR side
Method is used for quantitative two kinds of lactic acid bacteria culturers for making lucky scholar, includes Lactococcus lactis and Lactobacillus
Paracasei, design of primers, can be in amplifications after Successful amplification target dna using the tuf genes of target strain as amplification section
The melting curve (melting curve) of fragment (amplicon) obtains selectivity crest, rather than target dna can not then expand.
Actual result of implementation is, with the primer selectivity deficiency of tuf genes design, the DNA of non-targeted strain still has amplification phenomenon, can
To be detected by Real-time PCR instrument, the method must first extract upper real-time PCR boards analysis again after DNA,
It is expend analysis time and increase analysis cost more, and the quantitative bacterium number result of this analysis method is lack of consistency, such as
Lc.Lactis is higher than plate bacterium numbers with bacterium number quantitative qPCR, and Lb.paracasei is then plate bacterium numbers determines higher than qPCR
Measure bacterium number.
Document " Molecular quantification of lactic acid bacteria in fermented
milk products using real-time quantitative PCR.International Journal of Food
Microbiology 97 (2004) 197-207. " discloses a kind of real time quantitative PCR method and is used for absolute quantitation fermented dairy product
Lactic acid bacteria, comprising Streptococcus thermophilus, Lactobacillus delbrueckii, L.casei,
L.paracasei, L.rhamnosus, L.acidophilus and L.johnsonii, the survey from six commercial fermentation products
Examination, quantitative and traditional count of bacteria of mitogenetic technology do not have significant difference, and the still qualitative mirror of real time quantitative PCR method
Other lactobacillus inoculation, the detecting threshold values of its direct quantitative is 103Cell/mL.The method goes up real- again after must first extracting DNA
Time PCR boards are analyzed, expend analysis time and increase analysis cost more, and its primer is amplification 16S rRNA gene
Sequences regions, but because primer selectivity deficiency, non-targeted strain DNA can be also amplified.
Document " Quantitative Real-Time PCR Analysis of Fecal Lactobacillus
Species in Infants Receiving a Prebiotic Infact Formula.APPLIED AND
ENVIRONMENTAL MICROBIOLOGY, Apr.2006, p.2359-2365. " disclose and different bacterium are directed to 16S-23S regions
Compared with kind designs primer and probe sequence, but experimental result only sees detecting limit bacterium number relative with distinct methods, without
The quantitative data of lactic acid bacteria bacterium number.
Document " Quantification of total bacteria, enterobacteria and lactobacilli
populations in pig digesta by real-time PCR.Vet Microbiol.2006Apr 6；114(1-2):
165-70. " discloses an analysis method, it is necessary to upper real-time PCR boards analysis again after DNA is first extracted, when expending analysis more
Between with increase analysis cost.
Document " Development of a strain-specific real-time PCR assay for
enumeration of a probiotic Lactobacillus reuteri in chicken feed and
Intestine, PLOS One.2014Feb 27；9 (2) ", disclosing a kind of Real-time PCR methods can analyze in chicken intestinal
Lactobacillus reuteri viable counts, the method can only measure viable count.
Document " Advantageous direct quantification of viable closely related
probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions
by Propidium Monoazide--qPCR.PLOS One.2013Dec 17；8 (12) " disclose a kind of Real-time PCR
The method of quantitative probiotics bacterium number, and the PMA that arranges in pairs or groups (propidium monoazide) method distinguishes alive or dead probiotics.
The strain of analysis includes Lactobacillus acidophilus La-5, Bifidobacterium animalis
subsp.lactis Bb-12,_Lactobacillus sakei subsp.sakei 2a。
Document " Species-specific quantification of probiotic lactobacilli in
yoghurt by quantitative real-time PCR.J Appl Microbiol.2013Dec；115(6):1402-
10. ", this method designs primer with heat shock protein (HSP60) for target area, to identify and quantify different lactobacillus things
Kind, but above real-time PCR boards are analyzed again after first must extracting DNA from sample (yogurt), and it is expend analysis time and increasing more
Add analysis cost.
Document " Detection and quantification of probiotic strain Lactobacillus
Gasseri K7in faecal samples by targeting bacteriocin genes. " disclose a kind of method with
In Real-time PCR analysis health adult's excrement Lactobacillus gasseri K7 viable counts and find out in excrement whether
There is its bacterial strain to produce gassericin K7A or K7B, the method goes up real-time PCR machines again after must first extracting DNA
Platform is analyzed, expend analysis time and increase analysis cost more, and only detects viable count.
Document " Quantitative strain-specific detection of Lactobacillus
rhamnosus GG in human faecal samples by real-time PCR.J Appl
Microbiol.2009Feb；106(2):506-14. " discloses to be analyzed in health adult's excrement with Real-time PCR
Lactobacillus rhamnosus GG viable counts, the method go up real-time PCR boards point again after must first extracting DNA
Analysis, it is expend analysis time and increase analysis cost more, and only detect viable count.
Document " Representational difference analysis and real-time PCR for
strain-specific quantification of Lactobacillus sobrius sp.nov.Appl Environ
Microbiol.2005Nov；71(11):7578-81. " is disclosed one kind and analyzed with normal PCR and real-time PCR
The method of Lactobacillus sobrius sp.nov. strains, with RDA (Representational difference
Analysis) fragment as target gene and quantifies bacterium number.
Document " Quantification by real-time PCR of Lactococcus lactis
subsp.cremoris in milk fermented by a mixed culture.Appl Microbiol
Biotechnol.2005Jan；66(4):414-21. " discloses lactic acid bacteria (Lactococcus cremoris in analysis sample
ATCC 19257) viable count method, it is necessary to first from cheese samples extract DNA after upper real-time PCR boards analysis again, it is more
Analysis time and increase analysis cost are expended, and only detects viable count.
Document " Quantitative analysis of diverse Lactobacillus species present
in advanced dental caries.J Clin Microbiol.2004Jul；42(7):3128-36. " disclose a kind of use
The method of normal PCR and the lactobacillus species in real-time PCR analysis carious tooth sufferers oral cavity, sample must be subjected to DNA extractions
Afterwards, then upper real-time PCR boards are analyzed and quantified, expend analysis time and increase analysis cost more.
Document " Influence of Intrapartum Antibiotic Prophylaxis for Group B
Streptococcus on Gut Microbiota in the First Month of Life.J Pediatr
Gastroenterol Nutr.2016Feb；62(2):304-8. " discloses the pregnant woman that the culture of Type B streptococcus is positive, in
Preventive antibiotics are administered during production, the streptococcic probability of infection of newborn Type B can be avoided, but its enterobacteriaceae phase may be influenceed,
The microflora and quantity of enteron aisle in ewborn infant excrement are analyzed with real-time PCR boards, needs also exist for carrying out DNA extractions
Take, expend analysis time and increase analysis cost.
Document " Direct PCR of Intact Bacteria (Colony PCR) .Current Protocols in
A.3D.1-A.3D.6, May 2008. " discloses a kind of traditional PCR method to Microbiology, enters by specific dna sequence
The confirmation of row transition strain, it is not required to purifying extraction DNA, it is only necessary to which picking thalline is added in PCR reactants, sudden and violent after being pyrolyzed using thalline
The DNA of dew enters performing PCR amplification as template, and carries out band confirmation after colloid electrophoresis runs glue, is to identify transition strain
It is no to carry target gene, but this method race glue time is time-consuming and can not accomplish the function of quantitative bacterium number.
Summary prior art, existing probiotics and Analysis of pathogenic bacteria technology are detecting viable bacteria mostly, can not learn sample
Whether once by other strain pollutions, and first DNA extraction or extraction RNA is needed to be converted into cDNA, could be with PCR, Real-time
PCR is carried out qualitatively or quantitatively, and can not be extracted nucleic acid by testing sample (such as Yoghourt) and directly be carried out qualitative and quantitative analysis, except
Analysis time is expended with outside increase analysis cost, per one experimental procedure of increase more, increasing the machine of experimental error and pollution
Meeting, therefore, how to design and a kind of be not required to the probiotics and disease that first DNA extraction, RNA steps can be in qualitative and quantitative sample
The method of opportunistic pathogen, become the important topic of the invention to be solved herein.
The content of the invention
The technical problem to be solved in the present invention is to be directed to probiotics (heat goes viable bacteria) and pathogen thalline through hot processing of going out
Fungal counting, traditional selective medium culture can not be passed through and counted, lack on thalline is quantitative rationally, quick, tool is special
The detection method of different in nature (or selectivity), and the present invention applies real-time quantitative polymerase chain reaction (Real-time PCR),
Quantitative detecting method and the application of probiotics (heat goes viable bacteria) and pathogen thalline selectivity, its high sensitivity, specificity are provided
By force, stability is good, simple to operate, and energy quick detection goes out the bacterium number of probiotics (heat goes viable bacteria) and pathogen thalline in inspection product to be surveyed
Detection.
To solve the above problems, the purpose of the present invention is a kind of analysis of offer and quantifies probiotics and pathogen thalline quantity
Method, this method comprises the steps of：Step 1:An inspection product are taken to be dissolved in sterilized water, with high speed centrifugation, by thalline under,
Sterilized water dilution is added, as bacterium sample to be measured；Step 2:Bacterium sample to be measured is with real-time quantitative polymerase chain reaction (Real-
Time) PCR collocation specific primers group；The wherein specific primers group is the conservative gene with object bacteria, including 16S rDNA,
23S rDNA, 16S-23S rDNA interior transcriptional domain (internal transcribed spacer, ITS) or housekeeping gene
For (house-keeping genes) sequence fragment as amplification target, the wherein object bacteria is probiotics or pathogen；With amplification
The melting curve (melting curve) of fragment whether there is selectivity crest, determines whether the bacterium sample to be measured contains non-targeted bacterium
Kind；Step 3:Bring the inspection product Ct values of acquisition the regression equation of standard curve made from standard items into, bacterium to be measured can be learnt
The thalline quantity of sample.
For up to aforementioned invention purpose, the wherein probiotics is Bifidobacterium (Bifidobacterium), enterococcus spp
(Enterococcus), Lactobacillus (Lactobacillus), streptococcus (Streptoccus), but it is not limited to above-mentioned bacterium
Category.
Up to aforementioned invention purpose, the wherein Bifidobacterium (Bifidobacterium), to include bifidobacterium infantis
(B.infantis), lactic acid Bifidobacterium (B.lactis), bifidobacterium longum (B.longum)；The wherein enterococcus spp
(Enterococcus) comprising enterococcus faecalis (E.faecalis), VREF (E.faecium)；The wherein Lactobacillus
(Lactobacillus), comprising lactobacillus acidophilus (L.acidophilus), Lactobacillus casei (L.casei), lactobacillus fermenti
(L.fermentum), Lactobacillus paracasei (L.paracasei), Lactobacillus salivarius (L.salivarius)；The wherein streptococcus
Category (Streptococcus) includes streptococcus thermophilus (S.thermophilus), but is not limited to above-mentioned strain.
To include Aerononas punctata category (Aeromonas), bacillus up to aforementioned invention purpose, the wherein pathogen
(Bacillus), Enterobacter (Enterobacter), but it is not limited to above-mentioned Pseudomonas.
For up to aforementioned invention purpose, the wherein object bacteria specific primers group is the conservative gene with object bacteria, includes 16S
RDNA, 23S rDNA, 16S-23S rDNA interior transcriptional domain (internal transcribed spacer, ITS) have
The housekeeping gene (house-keeping genes) of specific function, the sequence such as including dnaK, fusA, groEL, gyrB, recA, tuf
Column-slice section is not limited to said gene as amplification target.
For up to aforementioned invention purpose, the wherein object bacteria specific primers length is 16~25bp, the piece of primer sets amplification
Segment length most preferably 50~150bp.
For up to the preferable circulation of aforementioned invention purpose, wherein real-time quantitative polymerase chain reaction (Real-time) PCR
Condition is：(1) 94~95 DEG C, 10 minutes；(2) 94~95 DEG C, 10~15 seconds；(3) 55~60 DEG C, 60~70 seconds；(4) 94~95
DEG C, 10~15 seconds；(5) 55~60 DEG C, 60~70 seconds (6) 94~95 DEG C, 10~15 seconds；Wherein impose a condition (2) and (3) repetition
30~45 circulations.
To be followed up to aforementioned invention purpose, wherein the optimal of the real-time quantitative polymerase chain reaction (Real-time PCR)
Ring condition is：(1) 95 DEG C, 10 minutes；(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6)
95 DEG C, 15 seconds；Condition (2)~(3) repeat 40 circulations.
For up to aforementioned invention purpose, the wherein specific primers group sequence is SEQ ID NO:1 and SEQ ID NO:2 is available
To expand Lactobacillus paracasei (L.paracasei) sequence fragment.
For up to aforementioned invention purpose, the wherein specific primers group sequence is SEQ ID NO:3 and SEQ ID NO:4 is available
To expand lactobacillus fermenti (L.fermentum) sequence fragment.
Up to aforementioned invention purpose, to be produced wherein inspection product include the food containing profitable probliotics, pharmaceuticals, dentifrice, chemical industry
Product, feed addictive, the dentifrice include toothpaste, mouthwash, tooth powder.
For that up to aforementioned invention purpose, should be analyzed with Protocols in Molecular Biology and quantify probiotics and pathogen thalline quantity
Whether method uses face including Analysis of pathogenic bacteria, chemical products in food or pharmaceuticals by pathogen contamination etc..
Another object of the present invention is a kind of analysis and quantifies the Real-time PCR of probiotics and pathogen thalline quantity
Set group is examined, the set group includes：SYBR Green qPCR Mix, the specific primers group for detecting object bacteria, the selectivity
Primer sets are the conservative genes with object bacteria, comprising 16S rDNA, 23S rDNA, 16S-23S rDNA interior transcriptional domain
(internal transcribed spacer, ITS) or housekeeping gene (house-keeping genes) sequence fragment conduct
Target is expanded, the object bacteria is probiotics or pathogen；For detecting probiotics and pathogen thalline number.
For up to aforementioned invention purpose, the wherein probiotics is Bifidobacterium (Bifidobacterium), enterococcus spp
(Enterococcus), Lactobacillus (Lactobacillus), streptococcus (Streptococcus), but be not limited to above-mentioned
Pseudomonas；Wherein the Bifidobacterium (Bifidobacterium) includes bifidobacterium infantis (B.infantis), lactic acid bifid bar
Bacterium (B.lactis), bifidobacterium longum (B.longum)；Wherein the enterococcus spp (Enterococcus) includes enterococcus faecalis
(E.faecalis), VREF (E.faecium)；Wherein the Lactobacillus (Lactobacillus) includes acidophilus breast bar
Bacterium (L.acidophilus), Lactobacillus casei (L.casei), lactobacillus fermenti (L.fermentum), Lactobacillus paracasei
(L.paracasei), Lactobacillus salivarius (L.salivarius)；Wherein the streptococcus (Streptococcus) includes thermophilic
Streptococcus (S.thermophilus), but it is not limited to above-mentioned strain.
To include Aerononas punctata category (Aeromonas), bacillus up to aforementioned invention purpose, the wherein pathogen
(Bacillus), Enterobacter (Enterobacter), but it is not limited to above-mentioned Pseudomonas.
For up to aforementioned invention purpose, the specific primers group is SEQ ID NO:1 and SEQ ID NO:2 can be used for amplification secondary
The sequence fragment of Lactobacillus casei (L.paracasei).
For up to aforementioned invention purpose, the wherein specific primers group sequence is SEQ ID NO:3 and SEQ ID NO:4 is available
To expand lactobacillus fermenti (L.fermentum) sequence fragment.
Brief description of the drawings
Fig. 1 is Lactobacillus paracasei (Lactobacillus paracasei) RT-PCR standard curves；
Fig. 2 is the melting curve (Melting curve) of the inspection product containing Lactobacillus paracasei (L.paracasei)；
Fig. 3 is the melting curve (Melting curve) of the inspection product containing lactobacillus acidophilus (L.acidophilus)；
Fig. 4 is the RT-PCR standard curves of lactobacillus fermenti (L.fermentum)；
Fig. 5 is the melting curve (Melting curve) of the inspection product containing lactobacillus fermenti (L.fermentum)；
Fig. 6 is the Real-time PCR amplification product fluorescence analysis figures of the toothpaste inspection product containing Lactobacillus paracasei；
Fig. 7 is the standard curve of the Bacillus oleronius inspection product through 10 times of serial dilutions.
Embodiment
The technical problem to be solved in the present invention is to be directed to probiotics (heat goes viable bacteria) and pathogen thalline through hot processing of going out
Fungal counting, it is general and traditional selective medium culture can not be passed through count, lack on thalline is quantitative rationally, soon
Fast, special detection method, and the present invention applies real-time quantitative polymerase chain reaction (Real-time PCR), there is provided through heat
Quantitative detecting method and the application for processing probiotics and the pathogen thalline selectivity of going out, its high sensitivity, high specificity, stability
Good, simple to operate, energy quick detection goes out the fungal counting of probio thalline (heat goes viable bacteria) and pathogen thalline in inspection product to be surveyed.
Specifically, the invention provides a kind of detection that probiotics and pathogen bacterium number are analyzed using Protocols in Molecular Biology
Method, the technical method taken comprise the following steps：
Step 1. pre-treatment：
1 ± 0.005 gram of bacterium standard items or the inspection product containing bacterium are weighed, are placed in sterile centrifugation tube, are added appropriate sterile
Water, standard items or inspection product bacterium powder are completely dissolved in sterilized water with oscillator.Entered with 10,000~11,000rpm, 4 DEG C of conditions
Row centrifugal operation 30 minutes, removes supernatant after the completion of centrifugation, add sterilized water by it is a small amount of repeatedly in a manner of back dissolving thalline, pour into conjunction
Quantified in suitable volumetric bottle, bacterium solution or inspection product bacterium solution to be measured are laid in as standard items.Standard items deposit bacterium solution can be through appropriate
Dilution is prepared into the standard items bacterium solution of five concentration respectively；Inspection product bacterium solution to be measured can also be diluted to debita spissitudo again, make its inspection product
Ct values can fall in standard curve range.
Step 2. real-time quantitative polymerase chain reaction：
Template amplification is carried out using the specific primer group of the probiotics contained by inspection product.In order to bacterial strain selectivity, draw
Thing is designed with the conservative gene of target probiotics and pathogen, includes interior turn of 16S rDNA, 23S rDNA, 16S-23S rDNA
Record area (internal transcribed spacer, ITS) or the housekeeping gene (house-keeping with specific function
Genes), sequence fragment is used as amplification target including dnaK, fusA, groEL, gyrB, recA, tuf etc., but is not limited to foregoing several
Kind gene.Related gene order refers to ncbi database.The primer group need to include preposition primer (Forward
Primer) and primer (Reverse primer) is inverted, primer length is 16~25bp, and the fragment length of primer sets amplification is optimal
For 50~150bp.
Real-time quantitative polymerase chain reaction reagent dosage refers to SYBR GreenERTM qPCR SuperMix for ABI
PRISM specifications are carried out.React the μ L of total amount 20：The μ L of SYBR Green qPCR Mix 10,5 μM of preposition primers and to invert primer each
1 μ L, DNA profiling (standard items bacterium solution or bacterium solution to be measured) 8 μ L.
Wherein, real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition, preferably impose a condition for：
(1) 94~95 DEG C, 10 minutes；(2) 94~95 DEG C, 10~15 seconds；(3) 55~60 DEG C, 60~70 seconds；(4) 94~95 DEG C, 10~
15 seconds；(5) 55~60 DEG C, 60~70 seconds (6) 94~95 DEG C, 10~15 seconds；Wherein impose a condition (2) and (3) repetition 30~45
Individual circulation.
Wherein, real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition, optimal is set as：(1)95
DEG C, 10 minutes；(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition
(2) 40 circulations, 4 DEG C of preservations~(3) are repeated.
After Successful amplification target dna, it can be obtained in melting curve (melting curve) analysis of amplified fragments single-minded
Property crest.
Step 3. is quantitatively with the processing of standard items bacterium powder and the drafting of standard curve：
By 109The quantitative of cell/more than g uses standard items bacterium powder, is handled through step 1 and is diluted to five concentration as mould
Plate, concentration range are preferably 105~108Cell/mL, real-time quantitative polymerase chain reaction (Real- is carried out according to step 2
Time PCR) Ct (Threshold Cycle) value is obtained, Ct values are inversely proportional with the template amount originated, dense with five different bacterium numbers
The logarithm (Log cells/mL) of degree is transverse axis, and its corresponding Ct value is depicted as standard curve, and obtain one time as the longitudinal axis
Return equation.And examine product bacterium solution and pass through step 2 real-time quantitative polymerase chain reaction (Real-time PCR), primer sets are single-minded
After ground is combined and expanded with object bacteria specific fragment, the inspection product Ct values of acquisition are brought the regression equation of aforesaid standards curve into
Formula, and it is multiplied by the quantity that target thalline in inspection product is learnt after extension rate.
The above method can analyze probiotics (heat goes viable bacteria) and the fungal counting of pathogen thalline through hot processing of going out, its
In, probiotics is Bifidobacterium (Bifidobacterium), includes bifidobacterium infantis (B.infantis), lactic acid bifid
Bacillus (B.lactis), bifidobacterium longum (B.longum)；Enterococcus spp (Enterococcus) includes enterococcus faecalis
(E.faecalis), VREF (E.faecium)；Lactobacillus (Lactobacillus), includes lactobacillus acidophilus
(L.acidophilus), Lactobacillus casei (L.casei), lactobacillus fermenti (L.fermentum), Lactobacillus paracasei
(L.paracasei), Lactobacillus salivarius (L.salivarius)；Streptococcus (Streptococcus) includes streptococcus thermophilus
(S.thermophilus), but it is not limited to above-mentioned strain.Wherein, pathogen includes Aerononas punctata category (Aeromonas), gemma
Bacillus (Bacillus), Enterobacter (Enterobacter), but it is not limited to above-mentioned Pseudomonas.
Embodiment 1：The quantitative detecting method of Lactobacillus paracasei (L.paracasei) in commercially available probiotics capsule product
Step 1. pre-treatment：
By the commercially available capsule product (strain deposit numbering CGMCC No.3247) containing Lactobacillus paracasei, capsule is taken out
In inspection product bacterium powder, weigh 1 ± 0.005 gram and be placed in sterile centrifugation tube, add appropriate amounts of sterilized water with oscillator will examine product bacterium
Powder is completely dissolved in sterilized water.With 10,000~11,000rpm, 4 DEG C of conditions carry out centrifugal operation 30 minutes, after the completion of centrifugation
Remove supernatant, add sterilized water by it is a small amount of repeatedly in a manner of back dissolving thalline, pour into suitable volumetric bottle and quantify, measure is examined
Product can be through being diluted to suitable multiple, and it is examined product Ct values can fall in the description of the drawings in Fig. 1 standard curve range.
Step 2. real-time quantitative polymerase chain reaction：
Using the specific primer group of Lactobacillus paracasei kind, the inspection product after pre-treatment are subjected to real-time quantitative polymerase company
Lock reactor.Target gene is 23S rDNA, and primer sets used are as follows, including preposition primer (Forward primer) and is inverted
Primer (Reverse primer)：Preposition primer (Forward primer)：5 '-TAG CGG CGA GCG AAG TG-3 ' (sequences
Arrange SEQ NO:1)；Invert primer (Reverse primer)：5 '-TCC TAC AAC CCC GAG AAG CA-3 ' (sequence SEQ
NO:2)；Above-mentioned primer sets expanding fragment length is 60bp.
Real-time quantitative polymerase chain reaction refers to SYBR GreenERTMQPCR SuperMix for ABI PRISM are said
Bright book is carried out.The μ L of reagent reacting total amount 20：The μ L of SYBR Green qPCR Mix 10,5 μM of preposition primers and invert each 1 μ of primer
L, DNA profiling (standard items or bacterium solution to be measured) 8 μ L.
Real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition is set as：(1) 95 DEG C, 10 minutes；
(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition (2) and (3) weight
Multiple 40 circulations, 4 DEG C of preservations.
Step 3. is quantitatively with the processing of standard items bacterium powder and the drafting of standard curve：
By 109Cell/more than g Lactobacillus paracasei (L.paracasei) thalline powder standard items, handled simultaneously through step a
Five concentration are diluted to as template, concentration range is preferably 105~108Cell/mL, carry out real-time quantitative according to step b and gather
Synthase chain reaction (Real-time PCR) obtains Ct values, is horizontal stroke with the logarithm (Log cells/mL) of five different bacteria concentrations
Axle, and its corresponding Ct value is depicted as the standard curve of Lactobacillus paracasei, and can obtain a regression equation as the longitudinal axis
Formula, as shown in figure 1, equation is Y=-3.3719X+43.433, R2=0.9964.
And product are examined after step b real-time quantitative polymerase chain reaction (Real-time PCR), by the inspection product of acquisition
Ct values bring the regression equation of aforesaid standards curve into, and are multiplied by after extension rate and draw the (heat of Lactobacillus paracasei in inspection product
Go viable bacteria) quantity, its testing result is as shown in Table 1.Other Fig. 2 show its melting curve (Melting curve), only
There is single crest, half melting temperature (Tm) all same of pcr amplification product, its product specificity can be confirmed；If the product of inspection are without pair
Lactobacillus casei, the negative control group (negative control) using lactobacillus acidophilus (L.acidophilus) as experiment, when
When being expanded using the Lactobacillus paracasei species specificity primer sets, high Ct values occur in experiment, represent amplified production
Without selectivity, single crest can not be presented in its melting curve, as shown in figure 3, it can thus be appreciated that this method really can effectively it is qualitative simultaneously
Quantify target strain.
The bacterium number quantitative result of Lactobacillus paracasei (L.paracasei) in one commercially available probiotics capsule of table
Embodiment 2：The quantitative detecting method of lactobacillus fermenti in probiotics powder product
Step 1. pre-treatment：
Probiotics powder product (the strain deposit numbering CGMCC of lactobacillus fermenti (L.fermentum) will be contained
No.3248), weigh 1 ± 0.005 gram to be placed in sterile centrifugation tube, add appropriate amounts of sterilized water and filled with oscillator by product bacterium powder is examined
Divide and be dissolved in sterilized water.With 10,000~11,000rpm, 4 DEG C of conditions carry out centrifugal operation 30 minutes, are removed after the completion of centrifugation
Supernatant, add sterilized water by it is a small amount of repeatedly in a manner of back dissolving thalline, pour into suitable volumetric bottle and quantify, determining inspection product can
Through being diluted to suitable multiple, it is examined product Ct values can fall in the description of the drawings in Fig. 4 standard curve range.
Step 2. real-time quantitative polymerase chain reaction：
Using the specific primer group of lactobacillus fermenti kind, the inspection product after pre-treatment are subjected to real-time quantitative polymerase chain
Reaction.Target gene is gyrB, and primer sets used are as follows, including preposition primer (Forward primer) and inverts primer
(Reverse primer)：Preposition primer (Forward primer)：5’-AAC GAA GAA ATC CGC TCC CTA T-3’
(SEQ ID NO:3)；
Invert primer (Reverse primer)：5’-CGT CAA AGT CGT CGC CAA A-3’(SEQ ID NO:
4)；Above-mentioned primer sets expanding fragment length is 61bp.
Real-time quantitative polymerase chain reaction refers to SYBR GreenERTMQPCR SuperMix for ABI PRISM are said
Bright book is carried out.The μ L of reagent reacting total amount 20：The μ L of SYBR Green qPCR Mix 10,5 μM of preposition primers and invert each 1 μ of primer
L, DNA profiling (standard items or bacterium solution to be measured) 8 μ L.
Real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition is set as：(1) 95 DEG C, 10 minutes；
(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition (2) and (3) weight
Multiple 40 circulations, 4 DEG C of preservations.
Step 3. is quantitatively with the processing of standard items bacterium powder and the drafting of standard curve：
By 109Cell/more than g lactobacillus fermenti (L.fermentum) thalline powder standard items, handled through step a and dilute
Five concentration are interpreted into as template, concentration range is preferably 105~108Cell/mL, real-time quantitative polymerization is carried out according to step b
Enzyme chain reaction (Real-time PCR) obtains Ct values, with the logarithm (Log cells/mL) of five different bacteria concentrations for transverse axis,
And its corresponding Ct value is depicted as the standard curve of lactobacillus fermenti as the longitudinal axis, and a regression equation can be obtained, such as schemed
Shown in 4, equation Y=-3.2555X+47.373, R2=0.9994.
And product are examined after step b real-time quantitative polymerase chain reaction (Real-time PCR), by the inspection product of acquisition
Ct values bring the regression equation of aforesaid standards curve into, and are multiplied by after extension rate and show that lactobacillus fermenti (go by heat in inspection product
Viable bacteria) quantity, its testing result is as shown in Table 2.Other Fig. 5 show its melting curve (Melting curve), only
Single crest, half melting temperature (Tm) all same of pcr amplification product, can confirm its product specificity.
The bacterium number quantitative result of lactobacillus fermenti (L.fermentum) in the probiotics powder product of table two
| Inspection product | Ct values | Real-time PCR count results (cell/g) |
| L.fermentum | 22.349 | 4.9×1011 |
| L.fermentum | 22.233 | 5.3×1011 |
Embodiment 3：Mix the quantitative detecting method of indivedual strains in probiotic products
Lactobacillus paracasei and lactobacillus fermenti containing same ratio, bacterium number are respectively individually in mixing probiotic products
5.6×1011Cell/g and 1.8 × 1011Cell/g, and probiotic products are mixed according to traditional light splitting luminance meter with wavelength
600nm detects bacterium number, and total bacteria count can only be learnt from bacterial cell concentration, and can not learn the bacterium number of indivedual strains, and this method
Expanded through the primer sets of strain selectivity, can accurately detect indivedual strain (secondary cheese breast bars in mixing probiotic products
Bacterium and lactobacillus fermenti) thalline quantity quantifies.
Step 1. pre-treatment：
Weigh 1 ± 0.005 gram to be placed in sterile centrifugation tube, it is with oscillator that inspection product bacterium powder is abundant to add appropriate amounts of sterilized water
It is dissolved in sterilized water.With 10,000~11,000rpm, 4 DEG C of conditions carry out centrifugal operation 30 minutes, are removed after the completion of centrifugation
Clear liquid, add sterilized water by it is a small amount of repeatedly in a manner of back dissolving thalline, pour into suitable volumetric bottle and quantify, determining inspection product can be through
Suitable multiple is diluted to, it is examined product Ct values can fall in Fig. 1 in the description of the drawings and Fig. 4 standard curve range.
Quantitative (the real-time quantitative polymerase chain reaction) of step 2A. Lactobacillus paracasei thalline quantities：
The back dissolving thalline obtained by step 1 is taken, using the specific primer group of Lactobacillus paracasei kind, by the inspection after pre-treatment
Product carry out real-time quantitative polymerase chain reaction.Target gene is 23S rDNA, and primer sets used are as follows, including preposition primer
(Forward primer) and invert primer (Reverse primer)：Preposition primer (Forward primer)：5’-TAG
CGG CGA GCG AAG TG-3’(SEQ ID NO:1)；Invert primer (Reverse primer)：5’-TCC TAC AAC
CCC GAG AAG CA-3’(SEQ ID NO:2)。
Real-time quantitative polymerase chain reaction refers to SYBR GreenERTMQPCR SuperMix for ABI PRISM are said
Bright book is carried out.The μ L of reagent reacting total amount 20：The μ L of SYBR Green qPCR Mix 10,5 μM of preposition primers and invert each 1 μ of primer
L, DNA profiling (standard items or bacterium solution to be measured) 8 μ L.
Real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition is set as：(1) 95 DEG C, 10 minutes；
(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition (2) and (3) weight
Multiple 40 circulations, 4 DEG C of preservations.
Step 3A. is quantitatively with the processing of standard items bacterium powder and the drafting of standard curve：
By 109Cell/more than g Lactobacillus paracasei (L.paracasei) thalline powder standard items, equally through pre-treatment simultaneously
Five concentration are diluted to as template, concentration range is preferably 105~108Cell/mL, real-time quantitative is carried out according to step 2A
Polymerase chain reaction (Real-time PCR) obtains Ct values, is with the logarithm (Log cells/mL) of five different bacteria concentrations
Transverse axis, and its corresponding Ct value is depicted as the standard curve of Lactobacillus paracasei, and can obtain a regression equation as the longitudinal axis
Formula.
And product are examined after step 2A real-time quantitative polymerase chain reaction (Real-time PCR), by the inspection of acquisition
Product Ct values bring the regression equation of Lactobacillus paracasei standard curve into, and are multiplied by after extension rate and draw secondary cheese in inspection product
The quantity of lactobacillus thalline (heat goes viable bacteria), its testing result is as shown in Table 3.
Quantitative (the real-time quantitative polymerase chain reaction) of step 2B. lactobacillus fermenti thalline quantities：
The back dissolving thalline obtained by step 1 is taken, using the specific primer group of lactobacillus fermenti kind, by the inspection product after pre-treatment
Carry out real-time quantitative polymerase chain reaction.Target gene is gyrB, and primer sets used are as follows, including preposition primer
(Forward primer) and invert primer (Reverse primer)：Preposition primer (Forward primer)：5’-AAC
GAA GAA ATC CGC TCC CTA T-3’(SEQ ID NO:3)；Invert primer (Reverse primer)：5’-CGT CAA
AGT CGT CGC CAA A-3’(SEQ ID NO:4)。
Real-time quantitative polymerase chain reaction refers to SYBR GreenERTMQPCR SuperMix for ABI PRISM are said
Bright book is carried out.The μ L of reagent reacting total amount 20：The μ L of SYBR Green qPCR Mix 10,5 μM of preposition primers and invert each 1 μ of primer
L, DNA profiling (standard items or bacterium solution to be measured) 8 μ L.
Real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition is set as：(1) 95 DEG C, 10 minutes；
(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition (2) and (3) weight
Multiple 40 circulations, 4 DEG C of preservations.
Step 3B. is quantitatively with the processing of standard items bacterium powder and the drafting of standard curve：
By 109Cell/more than g lactobacillus fermenti (L.fermentum) thalline powder standard items, handled through step 2B and dilute
Five concentration are interpreted into as template, concentration range is preferably 105~108Cell/mL, real-time quantitative polymerization is carried out according to step a
Enzyme chain reaction (Real-time PCR) obtains Ct values, with the logarithm (Log cells/mL) of five different bacteria concentrations for transverse axis,
And its corresponding Ct value is depicted as the standard curve of lactobacillus fermenti, and can obtain a regression equation as the longitudinal axis.
And product are examined after step 2B real-time quantitative polymerase chain reaction (Real-time PCR), by the inspection of acquisition
Product Ct values bring the regression equation of lactobacillus fermenti standard curve into, and are multiplied by after extension rate and draw acidified milk bar in inspection product
The quantity of bacterium thalline (heat goes viable bacteria), its testing result is as shown in Table 3.If with traditional light splitting luminance meter absorbing wavelength 600
(OD600) mode detects two kinds of bacterium mixed above, can only detect total bacteria count, cannot be distinguished by indivedual bacterium numbers, and the bacterium number degree of accuracy is not
It is good.Therefore, the method for quantitative bacterium number of the invention, the method used better than tradition.
Table three mixes the result that indivedual strain bacterium numbers are quantitative in probiotic products
Embodiment 4：The qualitative checking method of Lactobacillus paracasei in commercial toothpastes product
Step 1. toothpaste examines product pre-treatment：
By the commercially available toothpaste containing Lactobacillus paracasei, 5 ± 0.005 grams of toothpaste inspection product are weighed, are placed in sterile centrifugation tube,
Appropriate amounts of sterilized water is added inspection product are dissolved in sterilized water with oscillator.Carried out with 100~200g, under room temperature condition low speed from
The heart 1~2 minute, intermediate layer clarified solution is taken again with 200~300g room temperature conditions, carries out secondary low-speed centrifugal 1~2 minute, equally
After taking intermediate layer clarified solution, it is fitted into dialysis membrane and dialyses 24 hours, removes the interference factor that PCR reactions can be influenceed in toothpaste.Thoroughly
After the completion of analysis, inspection product of the clarified solution as real-time quantitative polymerase chain reaction (Real-time PCR) are drawn.
Step 2. real-time quantitative polymerase chain reaction：
Using the specific primer group of Lactobacillus paracasei kind, the inspection product after pre-treatment are subjected to real-time quantitative polymerase company
Lock reactor.Primer sets used are as follows, including preposition primer (Forward primer) and invert primer (Reverse
primer)：Preposition primer (Forward primer)：5’-TAG CGG CGA GCG AAG TG-3’(SEQ ID NO:1)；Instead
Put primer (Reverse primer)：5’-TCC TAC AAC CCC GAG AAG CA-3’(SEQ ID NO:2).
Real-time quantitative polymerase chain reaction refers to SYBR GreenERTMQPCR SuperMix for ABI PRISM are said
Bright book is carried out.The μ L of reagent reacting total amount 20：The μ L of SYBR Green qPCR Mix 10,5 μM of preposition primers and invert each 1 μ of primer
L, DNA profiling (standard items or bacterium solution to be measured) 8 μ L.
Real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition is set as：(1) 95 DEG C, 10 minutes；
(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition (2) and (3) weight
Multiple 40 circulations, 4 DEG C of preservations.
Step 3. real-time quantitative polymerase chain reaction (Real-time PCR) fluorescence analysis：
DNA of the inspection product containing Lactobacillus paracasei, can be from amplification product fluorescence point after real-time quantitative polymerase chain reaction
Analysis figure, as shown in fig. 6, find that inspection product and positive control group can produce fluorescence amplification curve (Amplification curve), can
Confirm the genetic fragment that Real-time PCR amplification product is Lactobacillus paracasei, thus the detection method can be applicable to toothpaste production
The qualitative detection of Lactobacillus paracasei in product.
The identification and analysis that environment contaminated bacteria monitors in fermentation process
If with the presence of contaminated bacteria fermentation tank may be caused to cause to lose groove because of pollution in fermentation process, cause to be produced into
This loss.This method is differentiated for possible pollution sources tracking, quickly reflected in earlier fermentation by real time aggregation enzyme chain reaction
Surely the presence or absence of Pseudomonas is polluted, to maintain the good quality of production.This embodiment is for Aerononas punctata category (Aeromonas), gemma
Pollution Pseudomonas is analyzed in the common water such as Bacillus (Bacillus) and Enterobacter (Enterobacter).
The inspection product pre-treatment of step 1. may be infected contaminated bacteria：
Inspection product (zymotic fluid, processing procedure water sample etc.) sampling 1mL is inserted in sterilized 1.5mL microcentrifugal tubes, with 10,
000~11,000rpm condition carry out centrifugal operation 3 minutes, and Aspirate supernatant is as inspection liquid A；Remove the sediment of supernatant
(pellet) 1mL aseptic deionized waters, are added, concussion is well mixed, is placed in after boiling water-bath boils 10 minutes, takes out centrifuge tube
To be cooled, equally with 10,000~11,000rpm conditions carry out centrifugal operation 3 minutes, and Aspirate supernatant is to another sterilized
1.5mL microcentrifugal tubes are as inspection liquid B.
Step 2. real-time quantitative polymerase chain reaction：
Inspection liquid after pre-treatment is all subjected to real-time quantitative polymerase chain reaction.Differentiate that specific primer group used is each
It is different, respective primer sets are separately added into, comprising preposition primer (Forward primer) and invert primer (Reverse
primer)：
(1) Aerononas punctata category (Aeromonas) is analyzed：Preposition primer (Forward primer)：5′-AAA GAG
GAY TAC AGC AAG AAG-3′(SEQ ID NO:5)；Invert primer (Reverse primer)：5′-GTC TTC ACT
TCG GAA GAR AC-3′(SEQ ID NO:6)。
(2) bacillus (Bacillus) is analyzed：Preposition primer (Forward primer)：5′-CCG TCA CAC
CAC GAG AGT TT-3′(SEQ ID NO:7)；Invert primer (Reverse primer)：5′-CAC CTT CCG ATA
CGG CTA CCT-3′(SEQ ID NO:8)。
Enterobacter (Enterobacter) is analyzed：Preposition primer (Forward primer)：5’–GAA GTT CTC
CTC RCA GAC CAA-3’(SEQ ID NO:9)；Invert primer (Reverse primer)：5’-GGG TTT TCC WGC
AGG TAK T-3’(SEQ ID NO:10)；
Real-time quantitative polymerase chain reaction refers to SYBR GreenERTMQPCR SuperMix for ABI PRISM are said
Bright book is carried out.The μ L of reagent reacting total amount 20：The μ L of SYBR Green qPCR Mix 10,5 μM of preposition primers and invert each 1 μ of primer
L, the μ L of DNA profiling (bacterium solution to be measured) 8.
Real-time quantitative polymerase chain reaction (Real-time PCR) cycling condition is set as：(1) 95 DEG C, 10 minutes；
(2) 95 DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition (2)~(3) weight
Multiple 40 circulations, 4 DEG C of preservations.
Step 3. real-time quantitative polymerase chain reaction (Real-time PCR) fluorescence analysis：
When inspection product contain the DNA of pollution Pseudomonas, after real-time quantitative polymerase chain reaction, its amplification product fluorescence analysis
Amplification curve in figure is in skew lines in logarithmic phase, genetic fragment of the Real-time PCR amplification product then containing pollution Pseudomonas,
It can confirm that the inspection product contain pollution Pseudomonas DNA；Ct value parts, Ct values are smaller, and fluorescent signal is more early by threshold value, represents contaminated bacteria
Category starting amount of DNA is the more；If there is Undetermined or high Ct values, as shown in the result of table four, representing may contaminated bacteria
Category starting amount of DNA is extremely low, and fluorescent signal can not be detected smoothly still under threshold value after 40 PCR cycles expand.And figure seven
It is observed that product are examined after 10 times of serial dilutions for environmental pollution bacterium Bacillus oleronius nutrient solutions, each bacterium number logarithm
There is highly linear between value and Ct values, 3.4 × 102During cfu/mL minimum bacteria concentration, using this method still be able to by
Smoothly detecting, it is known that minimum detecting limit can be situated between 102To 103cfu/mL.In addition to selectivity, this method has really in detecting
There is highly sensitive characteristic.
The monitored results of environment contaminated bacteria in the fermentation processing procedure of table four
【Biomaterial is deposited】
Domestic register information【Please according to deposit mechanism, date, numerical order annotation】
External register information【Please according to deposit country, mechanism, date, numerical order annotation】
The People's Republic of China (PRC), China General Microbiological culture presevation administration committee common micro-organisms center, it is deposited at
August in 2009 21 days, CGMCC No.3247
The People's Republic of China (PRC), China General Microbiological culture presevation administration committee common micro-organisms center, it is deposited at
August in 2009 21 days, CGMCC No.3248
Claims (17)
1. a kind of analyzed with Protocols in Molecular Biology and quantify probiotics and the method for pathogen thalline quantity, this method include down
Row step：
Step 1：Take an inspection product to be dissolved in sterilized water, with high speed centrifugation, by thalline under, add sterilized water dilution, as treating
Survey bacterium sample；
Step 2:Bacterium sample to be measured is with real-time quantitative polymerase chain reaction collocation object bacteria specific primers group；The wherein target
Bacterium specific primers group is the conservative gene with object bacteria, comprising 16S rDNA, 23S rDNA, 16S-23S rDNA interior transcription
As amplification target, the wherein object bacteria is probiotics or pathogen for area or housekeeping gene sequence fragment；
Selectivity crest is whether there is with the melting curve of amplified fragments, determines whether the bacterium sample to be measured contains non-targeted strain；
Step 3:Bring the inspection product Ct values of acquisition the regression equation of standard curve made from standard items into, bacterium to be measured can be learnt
The thalline quantity of sample.
2. one kind according to claim 1 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline quantity
Method, it is characterised in that the probiotics include Bifidobacterium, enterococcus spp, Lactobacillus, streptococcus.
3. one kind according to claim 2 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline quantity
Method, it is characterised in that the Bifidobacterium include bifidobacterium infantis, lactic acid Bifidobacterium, bifidobacterium longum；Wherein
The enterococcus spp includes enterococcus faecalis, VREF；Wherein the Lactobacillus includes lactobacillus acidophilus, Lactobacillus casei, hair
Kefir milk bacillus, Lactobacillus paracasei, Lactobacillus salivarius；Wherein the streptococcus includes streptococcus thermophilus.
4. one kind according to claim 1 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline quantity
Method, it is characterised in that the pathogen include Aerononas punctata category, bacillus, Enterobacter.
5. one kind according to claim 1 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline quantity
Method, it is characterised in that the specific primers group is the conservative gene with object bacteria, comprising 16S rDNA, 23S rDNA,
The sequence fragments such as 16S-23S rDNA interior transcriptional domain, housekeeping gene are as amplification target；Wherein housekeeping gene include dnaK,
fusA、groEL、gyrB、recA、tuf。
6. a kind of according to claim 1 or 5 analyzed with Protocols in Molecular Biology and quantify probiotics and pathogen thalline
The method of quantity, it is characterised in that the specific primers length is 16~25bp, the fragment length of primer sets amplification for 50~
150bp。
7. one kind according to claim 1 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline quantity
Method, it is characterised in that the cycling condition of the real-time quantitative polymerase chain reaction is：(1) 94~95 DEG C, 10 minutes；
(2) 94~95 DEG C, 10~15 seconds；(3) 55~60 DEG C, 60~70 seconds；(4) 94~95 DEG C, 10~15 seconds；(5) 55~60 DEG C, 60
~70 seconds (6) 94~95 DEG C, 10~15 seconds；Wherein impose a condition 30~45 circulations in (2) and (3) repetition.
8. one kind according to claim 1 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline quantity
Method, it is characterised in that the cycling condition of the real-time quantitative polymerase chain reaction is：(1) 95 DEG C, 10 minutes；(2)95
DEG C, 15 seconds；(3) 57 DEG C, 60 seconds (4) 95 DEG C, 15 seconds；(5) 55 DEG C, 60 seconds (6) 95 DEG C, 15 seconds；Condition (2)~(3) repeat 40
Individual circulation.
9. a kind of according to claim 1 or 5 analyzed with Protocols in Molecular Biology and quantify probiotics and pathogen thalline
The method of quantity, it is characterised in that the specific primers group sequence is SEQ ID NO:1 and SEQ ID NO:2, it can be used to expand
Increase the sequence fragment of Lactobacillus paracasei.
10. a kind of according to claim 1 or 5 analyzed with Protocols in Molecular Biology and quantify probiotics and pathogen thalline
The method of quantity, it is characterised in that the specific primers group sequence is SEQ ID NO:3 and SEQ ID NO:4, it can be used to expand
Increase the sequence fragment of lactobacillus fermenti.
11. one kind according to claim 1 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline number
The method of amount, it is characterised in that the inspection product include the food containing profitable probliotics, pharmaceuticals, dentifrice, chemical products, feeding
Feed additives, the dentifrice include toothpaste, mouthwash, tooth powder.
12. one kind according to claim 1 is analyzed with Protocols in Molecular Biology and quantifies probiotics and pathogen thalline number
The method of amount, it is characterised in that described to be analyzed with Protocols in Molecular Biology and quantify probiotics and the side of pathogen thalline quantity
Whether method applies to include in food or pharmaceuticals Analysis of pathogenic bacteria, chemical products by pathogen contamination.
13. a kind of analysis and the Real-time PCR inspection set groups for quantifying probio thalline and pathogen thalline quantity, the set group
Specific primers group comprising SYBR Green qPCR Mix, for detecting object bacteria；Wherein the specific primers group is with mesh
Mark bacterium conservative gene, comprising 16S rDNA, 23S rDNA, 16S-23S rDNA interior transcriptional domain or housekeeping gene sequence fragment
As amplification target, the wherein object bacteria is probiotics and pathogen；This inspection set group can be used for detection probiotics and pathogen
Bacterium number.
14. a kind of analysis according to claim 13 simultaneously quantifies the Real- of probio thalline and pathogen thalline quantity
Time PCR examine set group, it is characterised in that the probiotics includes Bifidobacterium, enterococcus spp, Lactobacillus, hammer
Pseudomonas；Wherein the Bifidobacterium includes bifidobacterium infantis, lactic acid Bifidobacterium, bifidobacterium longum；The wherein enterococcus spp
Include enterococcus faecalis, VREF；Wherein the Lactobacillus includes lactobacillus acidophilus, Lactobacillus casei, lactobacillus fermenti, pair
Lactobacillus casei, Lactobacillus salivarius；Wherein the streptococcus includes streptococcus thermophilus.
15. a kind of analysis according to claim 13 simultaneously quantifies the Real- of probio thalline and pathogen thalline quantity
Time PCR examine set group, it is characterised in that the pathogen includes Aerononas punctata category, bacillus, Enterobacter.
16. a kind of analysis according to claim 13 simultaneously quantifies the Real- of probio thalline and pathogen thalline quantity
Time PCR examine set group, it is characterised in that the specific primers group is SEQ ID NO:1 and SEQ ID NO:2 can be used for
Detect Lactobacillus paracasei.
17. a kind of analysis according to claim 13 simultaneously quantifies the Real- of probio thalline and pathogen thalline quantity
Time PCR examine set group, it is characterised in that the specific primers group is SEQ ID NO:3 and SEQ ID NO:4 can be used for
Detect lactobacillus fermenti.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108893549A (en) * | 2018-08-16 | 2018-11-27 | 山东探克生物科技股份有限公司 | Streptococcus thermophilus real-time fluorescence quantitative PCR primer pair and its application in a kind of fermented dairy product |
| CN110257485A (en) * | 2019-06-25 | 2019-09-20 | 吉林农业大学 | The fluorescence quantifying PCR method of compound probiotic ratio in a kind of detection fermented soybean milk |
| CN110804665A (en) * | 2018-08-06 | 2020-02-18 | 中国科学院微生物研究所 | Primer and method for identifying lactic acid bacteria in environmental sample at species level |
| CN111286550A (en) * | 2019-07-18 | 2020-06-16 | 大连民族大学 | Specific primer for amplifying lactobacillus paracasei and application thereof |
| CN111363835A (en) * | 2018-12-26 | 2020-07-03 | 中国科学院微生物研究所 | Method for identifying bifidobacteria in sample at species level and special primer thereof |
| CN111902546A (en) * | 2018-03-26 | 2020-11-06 | 巴克曼实验室国际公司 | Method for quantifying bioburden in a substance |
| CN112852931A (en) * | 2019-11-27 | 2021-05-28 | 特安康股份有限公司 | Method for analyzing beneficial bacterium strains and application of method in screening of beneficial bacterium strains |
| CN113981116A (en) * | 2021-11-30 | 2022-01-28 | 广州舒客实业有限公司 | Specific primer and kit for rapidly identifying lactobacillus salivarius and application of specific primer and kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509034A (en) * | 2008-11-18 | 2009-08-19 | 东华大学 | Human body intestinal canal flora detection parting and quantitative reagent kit |
-
2016
- 2016-06-21 CN CN201610455543.1A patent/CN107523607A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509034A (en) * | 2008-11-18 | 2009-08-19 | 东华大学 | Human body intestinal canal flora detection parting and quantitative reagent kit |
Non-Patent Citations (2)
| Title |
|---|
| HYUK-SANG KWON, ET AL.: "Rapid identification of probiotic Lactobacillus species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA", 《 FEMS MICROBIOLOGY LETTERS》 * |
| 赵文静 等: "实时荧光定量PCR技术在乳酸菌定量检测中的应用", 《食品与生物技术学报》 * |
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|---|---|---|---|---|
| CN111902546A (en) * | 2018-03-26 | 2020-11-06 | 巴克曼实验室国际公司 | Method for quantifying bioburden in a substance |
| CN110804665A (en) * | 2018-08-06 | 2020-02-18 | 中国科学院微生物研究所 | Primer and method for identifying lactic acid bacteria in environmental sample at species level |
| CN108893549A (en) * | 2018-08-16 | 2018-11-27 | 山东探克生物科技股份有限公司 | Streptococcus thermophilus real-time fluorescence quantitative PCR primer pair and its application in a kind of fermented dairy product |
| CN111363835A (en) * | 2018-12-26 | 2020-07-03 | 中国科学院微生物研究所 | Method for identifying bifidobacteria in sample at species level and special primer thereof |
| CN110257485A (en) * | 2019-06-25 | 2019-09-20 | 吉林农业大学 | The fluorescence quantifying PCR method of compound probiotic ratio in a kind of detection fermented soybean milk |
| CN111286550A (en) * | 2019-07-18 | 2020-06-16 | 大连民族大学 | Specific primer for amplifying lactobacillus paracasei and application thereof |
| CN112852931A (en) * | 2019-11-27 | 2021-05-28 | 特安康股份有限公司 | Method for analyzing beneficial bacterium strains and application of method in screening of beneficial bacterium strains |
| CN113981116A (en) * | 2021-11-30 | 2022-01-28 | 广州舒客实业有限公司 | Specific primer and kit for rapidly identifying lactobacillus salivarius and application of specific primer and kit |
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