CN101397586B - Composite gene chip for food-borne pathogenic bacteria detection - Google Patents
Composite gene chip for food-borne pathogenic bacteria detection Download PDFInfo
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Abstract
The invention relates to a composite gene chip for detecting food pathogenic microbes, which comprises eight parallel sub-arrays constructed by detection probe groups for detecting each microbe in the eight food pathogenic microbes, blank lines are respectively arranged among the eight sub-arrays to be as intervals, thus constructing a big array; the eight food pathogenic microbes are listeria monocytogenes, comma bacillus, vibrio parahaemolyticus, salmonella, shigella, staphylococcus aureus, campylobacter jejuni and escherichia coli. The gene chip can clearly and accurately detect the species of eight target food pathogenic microbes, and also can definitely exclude other off-target microbe samples out of the eight target food pathogenic microbes. Each food pathogenic microbe detection sub-array on the chip can clearly and definitely distinguish off-target microbe samples from target microbe samples.
Description
Technical field
The invention belongs to the food safety field, relate to the composite gene chip that food-borne pathogens detects.
Background technology
Food poisoning is meant have been taken in the food that contains biological, chemical hazardous and noxious substances or hazardous and noxious substances has been used as acute, the subacute disease that food is taken in non-infectious (not belonging to transmissible disease) that the back occurs.Food poisoning can be divided into bacillary, chemical, animality, vegetalitas, fungoid five big class poison according to the cause of disease material, wherein bacillary and chemical food poisoming is the most common.Bacterial food poisoning is meant that caused is the disease of main toxicity symptom with acute gastroenteritis owing to eat by after the food of pathogenic bacteria or its endotoxin contamination, and it is a modal class in the food poisoning.The common pathogen that causes bacterial food poisoning has: Vibrio parahemolyticus, Salmonellas, staphylococcus, escherichia coli, vibrio cholerae, Listeria monocytogenes, campylobacter jejuni, Bacillus proteus, bacillus cereus and Yersinia enterocolitica etc.In recent years, the food safety event frequency that causes of the food poisoning life that takes place frequently both at home and abroad threatens to human consumer's health.
How to find out the reason that causes food poisoning rapidly, be the important step of handling Public Health Emergencies; The food safety detection technique is the important technical that ensures food safety.In order to ensure people health better, personnel's injury, financial loss etc. are dropped to minimum, press for the Fast Detection Technique platform of setting up food poisoning, realize " initiatively ensureing " of food safety.Fast Detection Technique is the core sign of a safe and scientific development of state food, also is the important embodiment of a state food security control ability.Developed country such as America and Europe has dropped into substantial contribution the food safety detection gordian technique has been tackled key problems in recent years, and the detection technique level improves fast, and based on this standard is proposed more and more higher requirement, and restriction external product not up to standard enters.Therefore, the Fast Detection Technique of development food poisoning is the key that ensures China's food safety, the non-tariff technology barriers of reply trade, and meaning is especially great.
At present, " gold standard " method of both at home and abroad pathogenic bacteria being checked remains culture method.Need increase bacterium earlier behind the general collect specimen, positive findings appears and after, separate single bacterium colony again, carry out methods such as direct microscopy, separation and Culture, biochemical identification, serological test and experimentation on animals and could finally make a definite diagnosis.The cycle of these traditional methods is long, begins to calculate as increase the bacterium step from sepn process, and present qualification program needs more than five days; A little less than many bacterial classifications batch detection ability, be subject to the restriction that bacterium physiological condition and microbiotic use, and can not be used at present can't culturing bacterium or the evaluation of novel bacterial, be difficult to adapt to the internal and international situations of current fast development.Therefore, be necessary to develop one can be in enormous quantities, the detection methods of some kinds of microorganisms of rapid detection or pathogenic bacterium, food-borne pathogens is carried out active monitoring.The foundation of various in recent years Protocols in Molecular Biologies such as PCR, multiplex PCR, fluorescent quantitative PCR technique has greatly advanced detection and the evaluation of microorganism.But method high-throughout, that can identify great amount of samples simultaneously is still the difficult problem of pendulum in face of the investigator.
Biochip technology is a kind of high-new biotechnology that development in recent years is got up, and is a basic platform technology of life science.This technology is a kind of brand-new reverse solid-phase hybridization technology, with traditional be that the dot blot of medium is compared with the film, owing to used this rigidity hybrid vector of slide, DNA is bonded to the surface of glass slide of passing through activation treatment securely with the form of chemical bond, shows fabulous stability.The characteristics that this technology is outstanding are to detect simultaneously and identify multiple target gene, show fast, the incomparable advantages of other technology such as high-throughput, parallelization; Gene chip also has small, easy automatization of volume and advantage such as easy to operate, therefore now be widely used in the every field of life science, such as gene expression spectrum analysis, transgenation and polymorphism analysis, gene sequencing, new gene discovery, gene function analysis, genomic library mapping, disease prevention, medical diagnosis on disease, drug resistant gene detection, new drug development and evaluation, Toxicological Research and evaluation, bio-terrorism strick precaution, food safety and judicial expertise etc.
Biochip technology is used for the research that food-borne pathogens detects, is at home and abroad paid much attention to.Present food-borne pathogens detection chip can reduce two big classes according to the difference of probe, one class is the oligonucleotide probe chip, its probe length is generally 22-70mer, this type of chip probe is the pathogenic bacteria gene group sequence that has checked order by gathering, after special probe design and analysis software screening, use the dna synthesizer synthetic.Another kind of is the sub-probe chip of pcr amplification.By analyzing conservative or distinctive gene or the dna fragmentation on the pathogenic bacteria gene group checked order, design of amplification primers is carried out pcr amplification, is that amplicon about thousand bases of hundreds of to is as chip probe with the length that obtains.The food-borne causal agent detection chip of having reported so far, otherwise the bacterial classification that detects is single, either the gene that detects is single, or the purposes of detection chip is indeterminate, does not all embody the characteristics of gene chip high-throughput and high information content; And these researchs do not have tangible individual character and characteristic, all are that the identical or similar gene chosen is as the target gene that detects basically.
Summary of the invention
The composite gene chip that the object of the present invention is to provide a kind of food-borne pathogens to detect.
The composite gene chip that a kind of food-borne pathogens of the present invention detects, comprise eight parallel inferior arrays that make up with the detection probes group that detects each bacterium in eight kinds of food-borne pathogens, blank with delegation respectively between these eight inferior arrays as at interval, be configured to a big array;
Described eight kinds of food-borne pathogens are: Listeria monocytogenes (LM), vibrio cholerae (VC), Vibrio parahaemolyticus (VP), Salmonellas (Sal), Shigellae (Shi), streptococcus aureus (SA), campylobacter jejuni (CJ) and escherichia coli (EC);
Wherein, on the inferior array of Listeria monocytogenes 47 probes are arranged, sequence derives from 7 target genes shown in SEQ ID No.1-47; Have 93 on the inferior array of vibrio cholerae, sequence derives from 18 target genes shown in SEQ ID No.48-140; Have 33 on the inferior array of Vibrio parahaemolyticus, sequence derives from 9 target genes shown in SEQ IDNo.141-173; Have 31 on the inferior array of Salmonellas, sequence derives from 11 target genes shown in SEQID No.174-204; Have 31 on the inferior array of Shigellae, sequence derives from 8 target genes shown in SEQ ID No.205-235; Have 22 on the inferior array of streptococcus aureus, sequence derives from 9 target genes shown in SEQ ID No.236-257; Have 17 on the inferior array of campylobacter jejuni, sequence derives from 7 target genes shown in SEQ ID No.258-274; Have 73 on the inferior array of escherichia coli, sequence derives from 17 target genes shown in SEQ ID No.275-347;
Also comprise: 1 positive control probe, sequence is shown in SEQ ID No.348; 3 negative control probes, sequence are SEQ ID No.349-351.
Adopt the composite gene chip of food-borne pathogens detection of the present invention, detected the target food-borne pathogens of 8 kinds one by one.Detect Listeria monocytogenes, wherein the PPR (probe positive rate) of the inferior array report of LM is the highest, and mean P PR is 68.8%; Detect the Ying Nuoke listeria bacteria, the PPR of the inferior array report of LM is the highest, and mean P PR is 51.8%; Detect the listeria ivanovii sample, the PPR of the inferior array report of LM is the highest, and mean P PR is 59.6%; Therefore, the Preliminary detection data presentation, three different strains of this of listeria can make a distinction with this gene chip.Detect vibrio cholerae, the PPR of the inferior array report of VC is the highest, and mean P PR is 54.1%; Detect Vibrio parahaemolyticus, the PPR of the inferior array report of VP is the highest, and mean P PR is 66.7%; Detect Salmonellas, the PPR of the inferior array report of Sal is the highest, and mean P PR is 55.9%; Detect Shigellae, the PPR of the inferior array report of Shi is the highest, and mean P PR is 53.8%; Detect streptococcus aureus, the PPR of the inferior array report of SA is the highest, and mean P PR is 65.2%; Detect campylobacter jejuni, the PPR of the inferior array report of CJ is the highest, and mean P PR is 88.2%; Detect the crooked bacterium of large intestine, the PPR of the inferior array report of CJ is the highest, and mean P PR is 58.8%; There were significant differences with the PPR of the crooked bacterium of two different strain campylobacter jejunis of this chip detection generic and large intestine, can clearly two bacterial classifications be made a distinction fully.Detect intestinal bacteria, the PPR of the inferior array report of EC is the highest, and mean P PR is 47.9%.Above result shows that gene chip of the present invention is when detecting 8 target food-borne pathogens, and the PPR that object bacteria detects inferior array is significantly higher than other inferior array; Therefore this gene chip examining report result and actual detected sample meet fully.
The contriver also adopts gene chip of the present invention tentatively to detect except that the target food-borne pathogens of above-mentioned 8 kinds other certain representational biological sample, as non-object bacteria samples such as Lambda phage, people's gene group, streptococcus pneumoniae, swine streptococcus, streptococcus faecium, Pseudomonas aeruginosa and Neisseria meningitidiss; Found that, in these detected samples, except three gram-positive microorganisms of streptococcus are that streptococcus pneumoniae, swine streptococcus and streptococcus faecium are the PPR of the inferior array of LM is higher, when detecting all the other samples, the PPR of all inferior arrays is very low or do not have, and illustrates that the cross reaction of this chip and these non-object bacteria samples is very weak or does not have.Thereby, the specificity of the gene chip of the present invention of having given counterevidence.
Complete detection analysis by above-mentioned 18 kinds of known reference kind samples, tentative confirmation, the composite gene chip that food-borne pathogens of the present invention detects, can know correctly to detect 8 target food-borne pathogens kinds, also can get rid of 8 target food-borne pathogens kinds other non-object bacteria sample in addition clearly.And, show that according to the ordering of carrying out PPR behind 18 kind samples of this chip detection and comparative analysis each food-borne pathogens on this chip detects inferior array can distinguish object bacteria sample and non-object bacteria sample clear and clearly.
Description of drawings
Fig. 1 is the actual printing effect figure of the inferior array of last 8 Bacteria Detection of gene chip of the present invention (called after Biosafood-8 chip); The numerical value on each inferior array next door, right side shows the detection probes number of corresponding inferior array.
Fig. 2 is gene chip of the present invention (called after Biosafood-8 chip) Oligo microarray schematic layout pattern on chip.
Fig. 3 is the detected result scintigram that detects a bacterial strain 04LM233 of Listeria monocytogenes with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo left on figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Fig. 4 is after detecting three strain Listeria monocytogenes (LM) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Fig. 5 is the detected result scintigram that detects the bacterial strain V07-56 of vibrio cholerae with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo left on figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Fig. 6 is after detecting three strain vibrio cholerae (VC) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Fig. 7 is the detected result scintigram that detects the bacterial strain L-EP080 of Vibrio parahaemolyticus with gene chip of the present invention (called after Biosafood-8 chip).8 food-borne pathogens detect the position of inferior array on the arrow logo of the frame type to the right Biosafood-8 chip that vertically arrange on the figure left side; The frame type arrow logo left in figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Fig. 8 is after detecting three strain Vibrio parahaemolyticus (VP) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Fig. 9 is the detected result scintigram that detects the bacterial strain 0705YS0167 of Salmonellas with gene chip of the present invention (called after Biosafood-8 chip).8 food-borne pathogens detect the position of inferior array on the arrow logo of the frame type to the right Biosafood-8 chip that vertically arrange on the figure left side; The frame type arrow logo left in figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Figure 10 is after detecting three strain Salmonellass (Sal) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 11 is the detected result scintigram that detects the bacterial strain L-SH166 of Shigellae with gene chip of the present invention (called after Biosafood-8 chip).8 food-borne pathogens detect the position of inferior array on the arrow logo of the frame type to the right Biosafood-8 chip that vertically arrange on the figure left side; The frame type arrow logo left in figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Figure 12 is after detecting three strain Shigellaes (Shi) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 13 is the detected result scintigram that detects the bacterial strain 05ST028 of streptococcus aureus with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo left on figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Figure 14 is after detecting three strain streptococcus aureuses (SA) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 15 is the detected result scintigram that detects the bacterial strain 07C42 of campylobacter jejuni with gene chip of the present invention (called after Biosafood-8 chip).8 food-borne pathogens detect the position of inferior array on the arrow logo of the frame type to the right Biosafood-8 chip that vertically arrange on the figure left side; The frame type arrow logo left in figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Figure 16 is after detecting three strain campylobacter jejunis (CJ) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 17 is the detected result scintigram that detects the crooked bacterium of large intestine (CC) with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo left on figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Figure 18 is after detecting the crooked bacterium of a strain large intestine (CC) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 19 is the detected result scintigram that detects a bacterial strain EC882364 of escherichia coli with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo left on figure the right the sample of this chip detection, and the bacterial classification ownership of judging according to PPR.
Figure 20 is after detecting three strain escherichia colis (EC) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR average of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 21 is the detected result scintigram that detects the Lambda phage with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo that makes progress on figure the right the sample of this chip detection.
Figure 22 is after detecting a Lambda phage sample with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 23 is the detected result scintigram that detects people's complete genome DNA with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo that makes progress on figure the right the sample of this chip detection.
Figure 24 is after detecting people's complete genome DNA with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 25 is the detected result scintigram that detects a strain streptococcus pneumoniae (SP) with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo that makes progress on figure the right the sample of this chip detection.
Figure 26 is after detecting a strain streptococcus pneumoniae (SP) with gene chip of the present invention (called after Biosafood-8 chip), and the PPR that calculates according to the chip hybridization detection signal strength value of extracting from scintigram: the PPR to 8 inferior these samples of array detection on the chip compares.
Figure 27 is the detected result scintigram that detects a strain swine streptococcus (SS) with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo that makes progress on figure the right the sample of this chip detection.
Figure 28 is after detecting a strain swine streptococcus (SS) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 29 is the detected result scintigram that detects a strain streptococcus faecium (SF) with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo that makes progress on figure the right the sample of this chip detection.
Figure 30 is after detecting a strain streptococcus faecium (SF) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 31 is the detected result scintigram that detects a strain Neisseria meningitidis (NM) with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo that makes progress on figure the right the sample of this chip detection.
Figure 32 is after detecting a strain Neisseria meningitidis (NM) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 33 is the detected result scintigram that detects a Pseudomonas aeruginosa strain (PA) with gene chip of the present invention (called after Biosafood-8 chip).The arrow logo of frame type to the right that vertically arrange on the figure left side on the Biosafood-8 chip 8 food-borne pathogens detect the position of inferior array; A frame type arrow logo that makes progress on figure the right the sample of this chip detection.
Figure 34 is after detecting a Pseudomonas aeruginosa strain (PA) with gene chip of the present invention (called after Biosafood-8 chip), according to the PPR of the chip hybridization detection signal strength value calculating of extracting from scintigram; PPR to 8 inferior these samples of array detection on the chip compares.
Figure 35 is that mode that the PPR of 18 kind samples of Biosafood-8 chip detection detects inferior array according to 8 target food-borne pathogens is arranged relatively and gathered.
Embodiment
One, gene chip detects determining of target bacterial classification
The present invention intends selecting from above-mentioned bacterial classification 8 kinds of more common food-borne pathogens of Guangdong Province to be used to detect the development of gene chip, and they are respectively Listeria monocytogenes (LM), vibrio cholerae (VC), Vibrio parahaemolyticus (VP), Salmonellas (Sal), Shigellae (Shi), streptococcus aureus (SA), campylobacter jejuni (CJ) and escherichia coli (EC).
Sample source: all bacterial isolateses are all from bacterium kind storehouse, Guangdong Prov. Disease Prevention-control Center.
Below will detect gene chip called after " Biosafood-8 chip " at the oligonucleotide of these 8 kinds of food-borne pathogens developments.
Two, the design of oligonucleotide (Oligo) probe
89 detection target genes at 8 kinds of food-borne pathogens have designed 351 probes altogether, comprising 1 positive control probe and 3 negative control probes (table 4-1).At the gene chip detection probes of 8 kinds of food-borne pathogens designs, length all is 70mer, and other parameter is stablized Tm and GC% content etc. as annealing also all be according to design variable, carries out strictness control.The final probe sequence of determining and being used for chip manufacturing see the following form (table 1～table 10).
Integrated probe is summarized on the table 1 Biosafood-8 chip
For the certificate authenticity of quality control and data, positive control probe and the negative control probe parallel on chip array, have been designed with the sample detection probe.One of positive control probe, three of negative control probes.The design variable of their design variable and common sample detection probes is just the same, and for example, probe length is 70mer, the scope that annealing temperature and GC% content are also controlled in strictness.Their difference mainly is aspect the source.The 1# negative control is the human serum albumin gene fragment; 2# negative control behaviour hemoglobin gene fragment; The 3# negative control is continuously the artificial specified sequence " ATCG " of polyphone, does not have the biology encoding function; The positive control probe is and twice polyphone tumor-necrosis factor glycoproteins of the sample amplification labeled primer of restricted joint sequence coupling that adding part primer sequence to total length again at the two ends of this tumor-necrosis factor glycoproteins then is 70bp.
Table 2: totally 47 of the detection probes of process optimization improvement L.monocytogens (LM)
| SEQ?ID?No. | 5 '------probe sequence------3 ' |
| 1 | CCTGAAATCAGCGAACTTTACATCGTTGAGGGTGACTCAGCTGGTGGTTCAGCTAAACAAGGTCGTGACC |
| 2 | |
| 3 | GAAAAACAACCTTTACTTCGTAGAAGTAAATTGGTTAAGTTAGAAAGGGCGCACGGTGGATGCCTTGGCA |
| 4 | |
| 5 | TATAGCTCAGCTGGTTAGAGCGCACGCCTGATAAGCGTGAGGTCGATGGTTCGAGTCCATTTAGGCCCAC |
| 6 | GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACGGAGGAAGAGCTTGCTCTT |
| 7 | |
| 8 | GCGCATGCCACGCTTTTGAAAGATGGTTTCGGCTATCGCTTACAGATGGGCCCGCGGTGCATTAGCTAGT |
| 9 | |
| 10 | GCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGTCTTTTAA |
| 11 | GCAACCCTTGATTTTAGTTGCCAGCATTTAGTTGGGCACTCTAAAGTGACTGCCGGTGCAAGCCGGAGGA |
| 12 | CAAAGGGTCGCGAAGCCGCGAGGTGGAGCTAATCCCATAAAACTATTCTCAGTTCGGATTGTAGGCTGCA |
| 13 | GAGTTTGTAACACCCGAAGTCGGTAGGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATAATT |
| 14 | |
| 15 | GGATAGTATCCTTACGTGAATACATAGCGTGAGGAAGGCAGACCCAGGGAACTGAAACATCTAAGTACCT |
| 16 | AGAAAGTTATAAATGAAGCGGTCTGGAAAGGCCCGCCAAAGACGGTAACAGCCCGGTAGTTGAAATGGCT |
| 17 | |
| 18 | |
| 19 | |
| 20 | GGCCCTTCTCGGGTTACCGAATTCAGATAAACTCCGAATGCCATGTACTTATACTCGGGAGTCAGACTGC |
| 21 | |
| 22 | ATATCTAAGGTTTCCTGAGGAAGGCTCGTCCGCTCAGGGTTAGTCGGGACCTAAGCCGAGGCCGATAGGC |
| 23 | AGGGAATCGCACGAATGGAAATGTGCGTCCAAGCAGTGAGTGTGAGAAGTAGGCAAATCCGCTTCTCGCG |
| 24 | |
| 25 | TATAGGGGCTGACGCCTGCCCGGTGCTGGAAGGTTAAGAGGAGTGCTTAGCTTCGGCGAAGGTACGAATT |
| 26 | GAAGATGCAGGTTACCCGCGACAGGACGGAAAGACCCCGTGGAGCTTTACTGCAACCTGATATGGAATGT |
| 27 | GCTAGCATACGAGGAGGCAATGGTGGGATACTACCCTGGCTGTATGACCATTCTAACCCGCCACGCTTAG |
| 28 | GGATGGAAATCATTCGCAGAGTGTAAAGGCACAAGGGAGCTTGACTGCGAGACTGACAAGTCGAGCAGGG |
| 29 | |
| 30 | |
| 31 | GATGCATCTGCATTCAATAAAGAAAATTCAATTTCATCCATGGCACCACCAGCATCTCCGCCTGCAAGTC |
| 32 | |
| 33 | AGGATTGGATTACAATAAAAACAATGTATTAGTATACCACGGAGATGCAGTGACAAATGTGCCGCCAAGA |
| 34 | |
| 35 | ATGTTAATGAACCTACAAGACCTTCCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCT |
| 36 | AGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGTGTGGCGTATGGCCGTCAAGTTTAT |
| 37 | CTGGAGCGAAAACAATAAAAGCAAGCTAGCTCATTTCACATCGTCCATCTATTTGCCAGGTAACGCAAGA |
| 38 | ATTAATGTTTACGCYAAAGAATGCACTGGTTTAGCTTGGGAATGGTGGAGAACGGTAATTGATGACCGGA |
| 39 | |
| 40 | CAACAATCGCATCCGCAAGCACTGTAGTAGTCGAAGCTGGTGATACTCTTTGGGGTATCGCACAAAGTAA |
| 41 | CTGAAGTAAAACAAACTACACAAGCAACTACACCTGCRCCTAAAGTAGCAGAAACGAAAGAAACTCCAGT |
| 42 | AATGCTACTACACACGCTGTTAAAAGCGGTGACACTATTTGGGCTTTATCCGTAAAATACGGTGTTTCYG |
| 43 | CATGGGGTGGTAACGGACCAACTACATTTGATTGCTCTGGTTACACTAAATATGTATTTGCTAAAGCGGG |
| 44 | CTCCCTTCCACGTACTTCTGGCGCACAATACGCTAGCACTACAAGAATCTCTGAATCTCAAGCAAAACCT |
| 45 | TCTTTGACTATGGTAGCGGAATTTCTCACGTTGGTATCTACGTTGGTAATGGTCAAATGATTAACGCGCA |
| 46 | |
| 47 | GCGGTCAACTTTTAATCCTGACCTATGTGTATGGTAAAGAAACTCCTGATGGCATCAAGATTACACTGGA |
The detection probes of table 3:V.cholerae (VC), totally 93
| SEQ?ID?No. | 5 '------probe sequence------3 ' |
| 48 | AGCGGCAGCACAGAGGAACTTGTTCCTTGGGTGGCGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATTG |
| 49 | |
| 50 | GGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGTGGTTTGTTAAGTCAGATGTGAAAG |
| 51 | AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTTGGAGGTTG |
| 52 | GTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAAATCAGAATGT |
| 53 | TATCGTGATGAGTGTCCACACAGATTGATTCGGTTTAGATTAGAGAAGAGTATCTTAGTGTCCCGTTCGT |
| 54 | |
| 55 | CCTTCCTCCACAGGAAGTAAAACATGTGGGCGATTAGCTCAGTTGGGAGAGCACCTGCCTTACAAGCAGG |
| 56 | CGGTCAAGTGAATGAAGGAGTCGGTGCTGTTTCTTCTGTTACCGAAAGTGTAGGTAATTTAGTTGAGAAG |
| 57 | TCTTAATGCTGCAATTGAGGCTGCACGAGCTGGGGAAAATGGACGAGGTTTTGCTGTGGTTGCTGATGAG |
| 58 | ATTATGGCTTCGTCTGCTGCCGGCGATTAAGTCAGGCCGTCACATCATCACGAATGTGCGAGGCTTAAAC |
| 59 | |
| 60 | CCACGCCTAACATTGCCAAAGTGCACAACATGATAAGAGAGGCGGCGGAGATAGGGTATCGCCACTTTAA |
| 61 | ACAATCCAATTTTTACAGGGGGAAATGATGCAACTATCGAGTCAGAGCAATCCGAGCCTCAGTCAAAGGC |
| 62 | TGTCACTGTTGGTGATGAGCGTTATCGCCTCGTAGACAATTTGGACATTCCTTATCGTGGTCTATGGGCG |
| 63 | TTATATCGGGCAGATTCTAGACCTCCTGATGAAATAAAGCAGTCAGGTGGTCTTATGCCAAGAGGACAGA |
| 64 | |
| 65 | |
| 66 | |
| 67 | GACCTCCTGATGAAATAAAGCAGTCAGGTGGTCTTATGCCAAGAGGACAGAGTGAGTACTTTGACCGAGG |
| 68 | CATGCAAGAGGAACTCAGACGGGATTTGTTAGGCACGATGATGGATATGTTTCCACCTCAATTAGTTTGA |
| 69 | |
| 70 | AGATAGATATTACAGTAACTTAGATATTGCTCCAGCAGCAGATGGTTATGGATTGGCAGGTTTCCCTCCG |
| 71 | GCGATTGAAAGGATGAAGGATACCCTGAGGATTGCATATCTTACTGAAGCTAAAGTCGAAAAGTTATGTG |
| 72 | |
| 73 | GCGCAGAAGAAAGCAGCTTATGACCAATACGGCCATGCGGCATTTGAGCAAGGCGCGGGTGGTTTTGGTG |
| 74 | |
| 75 | TTGAAGAGTCTTGTGGCGGCGAAGCGGCGACGAAACACAAGCCGAAAGCCGAAGGCTTCTTTAATGGTGT |
| 76 | GGCAATAACTTGTACTGTGAAGTACCAGTGAGCTTCGCAATGGCGGCTCTGGGCGGTGAAGTAGAAGTAC |
| 77 | CAATCGGTGATGTCACACCTTTCTCATTCAAGGTGAGAGAGGTGTAGGCGCTACCGTCGCATTGATCACT |
| 78 | CGCAGCATCTTCCCTGATAAACAGAAAATCCCGACCGTGACTTATTTGAAAAGACAGATGAGTGATGAGA |
| 79 | |
| 80 | TGAAAGAATTATTGGCTCCTGTGCAGGCTTGGTGGCGAAGTGTCACCCCTCGTGAGCAAAAGATGGTAAT |
| 81 | TATGGTCTGTTATTTTCCACAGTGTTAATGACCAGCGTGGTCGTGCATTTGCCTGCCCAAGTGGCGCTTA |
| 82 | AGCGCCAAGGGGTGAGCGTGGATGCGATTGATATTGACCGTGGTAAAGTGAACGGCGTTGTGGAAGTCAA |
| 83 | CTGCCTGAAGGTTTAGAGCTCACTGGTATAGAGGGTACTCTGTGGCAAGGTCAAGCCGCGCAAGTTCGTT |
| 84 | |
| 85 | CGTACGTCCGCAAAGTCGACGTTAATCATGCCTGGGCGAGTAATCAGTTCAGCGATGCCTTGCACTGCAT |
| 86 | ATGTGTTCAACCGCGTTACCGCCGCCGCCACCCACTCCAACGACTTTGATTACCGCATCGTCAGACATTT |
| 87 | ATATTGCCACCAATCTGAATAACAGTACCAACGCTCGTTTTGCGCAGTGCCTGAGCATCAGTATTGATAC |
| 88 | TTCTTTATCTTCTAGTGCTGCATCACGACCCACTTGTGGATTCGCACCTGCACCTAGACCTTTGGTGATA |
| 89 | |
| 90 | CCAGTCCAACCCTGTTGGCAAATATCAATGAACCAAGTGGTGAAGCGGCGGATATTATTAGTCAAGTCGC |
| 91 | TGGAGCCAGTTATACGACGTTAGATGCCTATTTCCGTGAGTGGTCAACCGATGCGATTGCCCAAGATTAT |
| 92 | |
| 93 | CGTGGTGATTGTAATTCTCGGTATCTTGGCCGTCACAGCAGCGCCACGTTTCTTAAACCTGCAAGGCGAT |
| 94 | |
| 95 | ACCTCTAATAATCCTGCAACGGTTGCTATGCAGACTGGGGTTGCTGGAACTGCTTGTGGTAAACCAGCGC |
| 96 | CGGCAAGCCGATTTTTGGGGCCTTCCTCCTTTGCGGTGGTAACCACGCAGAGTGAAATATTAGCCTCACT |
| 97 | GTACGGGGTATTGTAATCGTTGGCTTTTGACCAGCAACGCCGCCATTCAAGTTTCTCCGCAGGCTATGCA |
| 98 | GTGTGGCGCTTTCCTTGGCGGGTTCGGGTGGGGCTTCTTTTCTTGATTTCGATAGTTTAGGCCGTGCTAC |
| 99 | |
| 100 | CTGACGGAATCAACCAAAGCGGTGACAAAGCAGGTTCAACCGTTTACAGCGCGAAAGGTACTTCTCTAGA |
| 101 | GAAGTTGGTGGCCGTGCTGAAGCTCGCCTATCTCTGAAAGATGGTAAGGCACAAGACAACTCTCGCGTAC |
| 102 | CTAGGCGTAAAAGCAAGCTACCGTTTTGCTGACCGTAACGCTGTTGACGCAATGGGTAATGTTGTAACTG |
| 103 | GGTGAAGATGGTTACTCTCTGTCTGCTATCTACACTTTCGGTGACACTGGCTTTAACGTAGGTGCTGGCT |
| 104 | TGCTAGCCGCTTCTTACCGCATGGAAAACCTGTACTTCGCAGGACTGTTCACTGACGGTGAGCTAGCGAA |
| 105 | CTATCGACGCAACTTACTACTTCAAGCCAAACTTCCGCTCTTACATCTCTTACCAGTTCAATCTGCTAGA |
| 106 | GGCTATCGGTCTACGTTACGACTTCTAATTGTTGACTTCAGGTCACACGCCAAACGCCTGCTCACTAGCA |
| 107 | CGGCCTAGCCGTACTTGCAGCCCTAAGCTCCGCTCCTGTATTTGCTCACCAAGAAGGTGACTTTATTGTG |
| 108 | GGTCCAATACTACTTTGGTGAAGCTAATTCGACTTTCCGTCCATATGTTGGTGCGGGTTTGAATTACACC |
| 109 | TGACAACATCAGTTTTGAAGTCCTCGCTGCTACGCCATTTTCACATAAGATTTCTACCTCTGGTGGTGAG |
| 110 | AACCTACAAAGCAGGTGCAGATGCCAAATCCACGGATGTTGAAATCAATCCTTGGGTATTTATGATCGCG |
| 111 | GGCTCTCGTGAATCTCGCATTTACTGGCGTATCTACAACAAGGCTGCTCAGCTTGGTTTAGATATGCACT |
| 112 | CGACGTTCTGCTCAATATCGAGGGGTATTTTGCAGGTTTGTGCGCGTACTCGGCCTCAATTATCAATTCC |
| 113 | CTGTACTTACGTCAATTGTGTGCACCAAAGTGTTTCTGTAATCCCTTGATTTGAAAACCTGTTACCAACC |
| 114 | ACTCCACACTAAGGCGGAGTTCTTGGATATTTTGGCAGCTTGTTTTTGCTGCTTAAATTTGGGCTACGCG |
| 115 | GTCACTGTACTTACGTCAATTGTGTGCACCAAAGTGTTTCTGTAATCCCTTGATTTGAAAACCTGTTACC |
| 116 | AGCTGATGAGGCAAAGAATCCTTTCACTGGTACAGCTATGGGGATTTTCTCATTTCCACGAAACTCTGCA |
| 117 | CGAATAAAGCATTCGCAATTACAGTCGGTGGCTTGACCCAAGCACAATGTAAGACTTTGGTTACAAGCGT |
| 118 | GCTGCTACTGGCGCTGGCGTAATTAAGTCCATTGCACCAGGAAGTGCCAACTTAAACCTAACTAATATCA |
| 119 | GGCCTCTGCCGTATTAGGGATGGCATTGGTCGCTGCTGGGAGTTATTACAAGCGGGAAGCTGAACTCATG |
| 120 | GGGTATTATGGGTGTGGTCTCAGCGGGTGTTGTTACGCTGGCTCAGCGTGCGATTGATTCGCAGAATATG |
| 121 | AGCTAGCTAATGGTTTGGTCAGCCTTGGTAAGGTTTCAGCTGATGAGGCAAAGAATCCTTTCACTGGTAC |
| 122 | GCTACTGGCGCTGGCGTAATTAAGTCCATTGCACCAGGAAGTGCCAACTTAAACCTAACTAATATCACGC |
| 123 | GGCCTCTGCCGTATTAGGGATGGCATTGGTCGCTGCTGGGAGTTATTACAAGCGGGAAGCTGAACTCATG |
| 124 | TGCAATGTATCCAGCCACGCCGAAAACTCGGTGTTATAGCGTTCACCCAGTAATTTGACACTCTGTGCCC |
| 125 | TAGCCCCAGCGATAAATGCGGTTCGTATCATTCGAGTAGGTGGTGGCAAACACATCAGAAAGGCTCAATT |
| 126 | CAGCTTAACCGCCGCTTGGTAGCCCTGCTTATAATGCATATATTCCGCGAAACCTTCTAACCACCACACA |
| 127 | CTCATGCTCTAAATTGAGAATCGACAAATCCGCATCATTGGCATAACGATAGGCGACAAAACGGGCTTGA |
| 128 | CAATCACTTGTCGCATCACATGACGAGCCTTTTGCTTGGTAATGGCATCTGGGCTAATCAATAAGCGGCC |
| 129 | ATGGCCAGACATCGCACGGAATAAGTTATTTAAACCATCGACCCATTGTGTATCTTGCGCCGTTTCTCGA |
| 130 | ACGTTATCCACCATTAAACTTAGGCTCTTCATTGCGCCCACCTGTTCACGGCCTTGGTCAAAAGCATGCG |
| 131 | GTAATACGCTGCGCGAATGAACTCACCATAGTTTTCCAAGCGACGAGCTTGCTGCGCTTCACCAGTATAG |
| 132 | GAGAAGCTTCGCTATAGAGGTTATCCAAGGTTGCCGTTGGTGCGTAAAACCATGAGTTGTAACAAGCTTG |
| 133 | GAGTAAGAAAGACCAGCGATGACGCACGCCCAAGCAATGGGGTGATGTTGGATACGTTGTTGAAAAGACA |
| 134 | CACATAACTTTTCGACTTTAGCTTCAGTAAGATATGCAATCCTCAGGGTATCCTTCATCCTTTCAATCGC |
| 135 | CGGAGGGAAACCTGCCAATCCATAACCATCTGCTGCTGGAGCAATATCTAAGTTACTGTAATATCTATCT |
| 136 | GGGAGTATGGAATCCCACCTAAAGCAGAAACTTCTTGTTCATCTGGATGAGGACTGTATGCCCCTAATAC |
| 137 | GGGCACTTCTCAAACTAATTGAGGTGGAAACATATCCATCATCGTGCCTAACAAATCCCGTCTGAGTTCC |
| 138 | TCTGTCCTCTTGGCATAAGACCACCTGACTGCTTTATTTCATCAGGAGGTCTAGAATCTGCCCGATATAA |
| 139 | GTCTACGAGGCGATAACGCTCATCACCAACAGTGACAAAACCATCTTGGACACAAAGCCGACCAATACAA |
| 140 | GCGCGGTTAAAGTGGCGATACCCTATCTCCGCCGCCTCTCTTATCATGTTGTGCACTTTGGCAATGTTAG |
The detection probes of table 4:V.parahaemolyticus (VP), totally 33
| SEQ?ID?No. | 5 '------probe sequence------3 ' |
| 141 | GGCCTAACACATGCAAGTCGAGCGGAAACGAGTTATCTGAACCTTCGGGGAACGATAACGGCGTCGAGCG |
| 142 | CGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGTGGTTTGTTAAG |
| 143 | CCTGGACAGATACTGACACTCAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA |
| 144 | ACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGA |
| 145 | AAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATA |
| 146 | GACAAACACCAAAATAACACATTCAAGTGTTCTTGGAATTTGAGTCCGGCAAAATCGAGTCTGCATCATG |
| 147 | GGTTTATAAAGTAAAGAGAAGAAGAGTTCCCAAACTCTTCAATCCAGTGTCCCGTTCGTCTAGAGGCCTA |
| 148 | TCGTCTAGAGGCCTAGGACACCGCCCTTTCACGGCGGTAACAGGGGTTCGACTCCCCTACGGGATACCAT |
| 149 | GACAAACACCAAAATAACACATTCAAGTGTTCTTGGAATTTGAGTCCGGCAAAATCGAGTCTGCATCATG |
| 150 | AAACGTCTGCGTGAGCTATCGTTCTTGAACTCAGGCGTTTCTATCAAGCTTATTGATGAGCGCGAAGCGG |
| 151 | GGTGGTATTCAAGCGTTCGTTCAGCACTTAAACACCAACAAAACACCAATCATCGAGAAAATCTTCCACT |
| 152 | TTCTGTTTCACCAACAACATTCCACAGCGCGATGGTGGTACTCACCTTGCTGGTTTCCGTGCGGCACTAA |
| 153 | GAACTATACATTGTGGAGGGTGACTCTGCGGGTGGTTCAGCTAAGCAGGGTCGTAATCGTAAGAATCAGG |
| 154 | GATCTATCGACTGTTGGTCGTATGAAGTTTAACAGCTCTATCGGTCGTGAAGATGCTCAAGAGCAAGGCA |
| 155 | CATGGACCAAAACAACCCGTTATCAGAAGTAACGCACAAGCGTCGTATTTCTGCATTGGGTCCTGGCGGT |
| 156 | CCTATCTGCAATCGAAGAAGGCCAATTCGTTATCGCTCAGGCGAACGCTAAGCTAAACGAAGATGGTACT |
| 157 | GCTCAGTACATGGACGTTGCGACAAACCAGGTTGTATCTATCGCTGCATCGCTTATCCCGTTCCTAGAAC |
| 158 | GYTGACATCCTACATGACTGTGAACATTAATGATAAAGACTATACAATGGCAGCGGTGTCTGGCTATAAG |
| 159 | GATCCTTGTTTGGACATCAACCGCTCATCGTCTGTCGATTACATGTACACCCACGCATTGCGCTCTGAGT |
| 160 | CCAACAACAGCAACTCACCGCTATGTTGCAGAGAAAATGCTAGAAAGTAGCAACAACTTAGCCGAGTACC |
| 161 | GCACGCAAGAAAACCAAACCTATACCTATGTTCGCTGTTGGTATCGCACCAGCTACTCGAAAGATGATCC |
| 162 | AACAAAGTGGTTGCACTCGGTGACAGCTTGTCTGATACAGGCAACATCTTTAACGCATCACAATGGCGCT |
| 163 | ATTGCCAAAGCGAAGAACCTTCCGCTCTACAACTGGGCAGTTGGCGGCGCGGCTGGTGAGAACCAATACA |
| 164 | GTACTTAACCTACGCAAAACTGGCGAAGAACTACAAACCAGCAAACACCTTGTTTACGCTTGAGTTTGGT |
| 165 | GTACTGTTTCCAGTTCGTTGTTTTGTAGCGAGGTTTCGGCATGAGACTAGGGGAATTAACTTACTTAGCC |
| 166 | TTTACCGTCATATAGGCGCTTAACCATTTTGAGCCTGAAGTCGTGAAAATAGATTGACCGTGAACGCTTT |
| 167 | GTAATTCGCTCGCTGACCAACAAAGCGGCAACGAAGTTGTACGATTAGGAAGCAACGAAAGCCGTATACT |
| 168 | AACGAGCTTCACGAGTTTGTTTGGCGTGAGCAAGGTTTTGAGGTGGATGACTCAAGCCTGACTCAAGCGA |
| 169 | TGCCTTCTATTGAGCAGTGCATTGAACGCTACGTTAAGCACCATGCAGAAGACTCGTTACCAGTGGAAGT |
| 170 | GCTGATTTTGAACTACATTCATGACAGCAACCACTCGTATGAGAACGTGACATTGCGTATTTTCGCAGGT |
| 171 | AGTGGTTGGTTGTACTGGGGCAGTGACCTTAAAGTTGAGCAAGTGCTTACATCAAATGAATGGCAGTCAA |
| 172 | CCAACAGACATCTGCAAATAAAGGAGGCCAGCATGAAGATTAAAGTAGCATCTGCGGTTTTGGCCGTATC |
| 173 | GGCGATTACATTCGCGTGGCAAACATCAAACTGTTCGCACAAGGCTCGACGGCTGAATCGACAATTAATA |
The detection probes of table 5 Salmonella spp. (Sal), totally 31
| SEQ?ID | 5 '------probe sequence------3 ' |
| No. | ? |
| 174 | GCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAAC |
| 175 | GCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAG |
| 176 | TGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT |
| 177 | AGTAGCGGCGAGCGAACGGGGAGGAGCCCAGAGCCTGAATCAGCATGTGTGTTAGTGGAAGCGTCTGGAA |
| 178 | CAGGTTTACCTGTGTGACTGCGTACCTTTTGTATAATGGGTCAGCGACTTATATTCTGTAGCAAGGTTAA |
| 179 | GGGTGAAAGGCCAATCAAACCGGGAGATAGCTGGTTCTCCCCGAAAGCTATTTAGGTAGCGCCTCGTGAA |
| 180 | GCTAACGTCCGTCGTGAAGAGGGAAACAACCCAGACCGCCAGCTAAGGTCCCAAAGTCATGGTTAAGTGG |
| 181 | CTGTGAAGATGCAGTGTACCCGCGGCAAGACGGAAAGACCCCGTGAACCTTTACTATAGCTTGACACTGA |
| 182 | CACGAAGGTTGGCTAATCCTGGTCGGACATCAGGAGGTTAGTGCAATGGCATAAGCCAGCTTGACTGCGA |
| 183 | GCTGTTCGCCATTTAAAGTGGTACGCGAGCTGGGTTTAGAACGTCGTGAGACAGTTCGGTCCCTATCTGC |
| 184 | TACGAGAGGACCGGAGTGGACGCATCACTGGTGTTCGGGTTGTCATGCCAATGGCACTGCCCGGTAGCTA |
| 185 | AACCTCTACAGGCTTGTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCGGTGGTTCAAGT |
| 186 | AACTGCCTTCAGGTTCAGAACTCTCCCTGTACGACATCGCTCCAGTGACTCCCGGTGTGGCCGTTGATTT |
| 187 | AGGCGTGGTGGAATGCGCCTATGTGGAAGGCGAYGGTCAGTATGCCCGTTTCTTCTCTCAGCCGCTGCTG |
| 188 | AGCTCTGCTGGTGGCGGGCGCAGCGAATGCGGCTGAAATTTATAATAAAGACGGCAACAAATTAGACCTG |
| 189 | GTAAAGTTGATGGCCTGCACTACTTCTCTGACGACAAAGGCAGCGACGGCGACCAGACCTACATGCGTAT |
| 190 | CGTTTGCGGGTCTGAAATTCGCTGACGCAGGTTCCTTCGATTATGGTCGTAACTACGGCGTAACCTATGA |
| 191 | GTTCTGCCGGAGTTCGGCGGCGACACCTACGGCGCTGACAACTTTATGCAGCAGCGTGGTAACGGCTATG |
| 192 | TACACCGGCGGCCTGAAATACGATGCGAACAACATCTATCTGGCAGCGCAGTATTCTCAGACCTATAACG |
| 193 | GTCCACCTCTTACGGTTTTGCCAACAAAGCGCAGAACTTTGAAGTGGTTGCTCAGTACCAGTTCGACTTT |
| 194 | TCAGCAAAGATTCGCTGATGCTTCAGCTRTATCCGGATGCGGAACTGCGGGAAAGYCATACCATTGATGT |
| 195 | CACGACTTACATCCTACTTCGAAAGTCGACATACTGTGATTGGCTTAGCAAGGAAGAGGAACAATGAAGC |
| 196 | TGGCTGGTATGGCGAAGTTTTACTCTCTTACGGGCGTCAGGTGATATTCCTGGCGTTGGCTAAGGGGTAA |
| 197 | TACCTTCAAATATGACGCTTAACGGCGTCGCATTGCTGCTTTCTATGTTTGTTATGTGGCCAATAATGCA |
| 198 | CATGAATGGCTGAGCGAGTCTGGTAATGGGGAATGATATCTCATTAATTGCCTTACTGGCATTTTCCACC |
| 199 | TGCAGCAGACACCGGACAATGGCACGAACCTGAGCAGTTCCGCYGTCGGCGGTATTCAGGGACAGGCAGA |
| 200 | AATACCGATGTGTCGCTGGAAGATATTATCAGCGAGAGCAGCAYTGACTGGCCGTGGTTCCTGAGCAACT |
| 201 | AGTATGCGCCCTTTGGCCTGAGCGTGCTGCCGGTGACCATTACGAAGAACTGCCGCAGTGTTAAGGATAT |
| 202 | GGTTATCACCTGCTGCACCAGGAAAGCATTAAGTTGATAGAACACCWACCCGCTTCTCTCTTGCAAAACA |
| 203 | TTAAGCGAACGTGTTTCCGTGCGTAATATGAAATTAATTATGGAAGCGCTCGCATTGTGGGCGCCAAGAG |
| 204 | GCATCATTATCATCTTTGTGAACTTTATTGGCGGTATTTCGGTGGGGATGACCCGCCATGGTATGGATTT |
The detection probes of table 6 Shigella spp. (Shi), totally 31
| SEQ?ID?No. | 5 '------probe sequence------3 ' |
| 205 | TGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGG |
| 206 | GATAACTACTGGAAACGGTAGCTAATACCGCATAAYGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTC |
| 207 | CGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGA |
| 208 | AACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGC |
| 209 | CTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCAC |
| 210 | AGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTC |
| 211 | AGCTGGAAAGTTGCAGTAATCCACTTTCAACAAACCGCACCAATGTCCAGGCACGGGTTAATGCCAGCAG |
| 212 | ACAGCCTGTACTCTCTGTTCATCCAGCAGTTGTGGGATAATATCGGCAGGATTCTGGGAAAGTTTACGTC |
| 213 | TCGTCTGGTGGCTGTCAAAACGGAAGTGACAAACCAGTTGATTGGTTTCTGCCAGCTTAACCATTTGCGG |
| 214 | GACAAGTTGATGTCATAAATGTGTTTCAGCAACTCGGAGGTATGCATTATTCCCACCCAGAATAACCAAC |
| 215 | ACTTCTTCCAACCCCTCTCATGCATCTAGAACTAAGCTACGGATTAACTCTGATATTATGATTAGCGCAG |
| 216 | GGTGAGGCTGTTACAGGAGACAATCTGTTTATAATAAATGGAGAAATTATTTCAGGTGGACATGGTGGCG |
| 217 | AGGGACTATTTCCGGTGGTAATGGAGGTAACAATTATGGTGAGGGTGATGGCGGTAATGGAGGTGATGCC |
| 218 | ATAAGGGCACGTTCGCTGGAGGCAACGGAGGTGCTGCTTACGGTTATGGTTATGATGGCTACGGTGGTAA |
| 219 | TCAAACACAGCATCTGCTGAACAGCTATCAACCCAGCAGAAATCATTAACCGGACTTGCCAGTGTTACTC |
| 220 | CCTCTTGCCAAAATATTGGCTTCCACTGAGCTTGGAGACAATACTATCCARGCTGCAAATGATGCAGCTA |
| 221 | GATAATGATACCGGCGCTCTGCTCTCCCTGGGCAGGGAAATGTTCCGCCTCGAAATTCTGGAGGACATTG |
| 222 | TTTTGTGGATGAGATAGAAGTCTACCTGGCCTTCCAGACCATGCTCGCAGAGAAACTTCAGCTCTCCACT |
| 223 | ACTGCCGTGAAGGAAATGCGTTTCTATGGCGTGTCGGGAGTGACAGCAAATGACCTCCGCACTGCCGAAG |
| 224 | TATGAAGCGATGGTCCTGATGGCGCAGCCGTTCTCTTACCGTTATCCGCTGGTTGATGGTCAGGGGAACT |
| 225 | ATCCGAAATCGTTCGCGGCAATGCGTTACACCGAATCCCGGTTGTCGAAATATTCCGAGCTGCTATTGAG |
| 226 | CCAAACTTCGACGGCACTTTGCAGGAGCCGAAAATGCTACCTGCCCGTCTGCCAAACATTTTGCTTAACG |
| 227 | |
| 228 | |
| 229 | ATCTACAATAGACATCGAGCCGGAAGGTGATGTTTACTTTCCTGAAATCCCCAGCAATTTTAGGCCAGTT |
| 230 | TGAACTATCTACCAGAAGTGTGAGCCCCTACCGGAAGGATTACATCTCGGATGATGACTCTGATGAAGAC |
| 231 | TCGAACACATTGTTGTGTCGCACACGCACCGAGGCAAAGGAGTCGCGCACAGTCTCATCGAATTTGCGAA |
| 232 | ACAATGTACCTGCCTGCAATTTGTACGCAAAATGTGGCTTTACTCTCGGCGGCATTGACCTGTTCACGTA |
| 233 | CCTCAAGTCTCGAACGAAACAGCGATGTACTGGTACTGGTTCTCGGGAGCACAGGATGACGCCTAACAAT |
| 234 | TAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTCACGCCGTATGTTATT |
| 235 | GGCAACCGGGTGAAGGTTATCTCTATGAACTGTGCGTCACAGCCAAAAGCCAGACAGAGTGTGATATCTA |
The detection probes of table 7 S.aureaus (SA), totally 22
| SEQ?ID?No. | 5 '------probe sequence------3 ' |
| 236 | GAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAAT |
| 237 | TGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGG |
| 238 | TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACAACTCTA |
| 239 | TAGTTGCCATCATTAAGTTGGGCACTCTAAGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGAC |
| 240 | GGGCTACACACGTGCTACAATGGACAATACAAAGGGCAGCGAAACCGCGAGGTCAAGCAAATCCCATAAA |
| 241 | CTGACATTCGGCGACCACGATGTACTTGACTATCGAACGGTCCAAAACCTTCTTGTCTAAATCCATTCAC |
| 242 | AACAGTGCGCAAAATCCCAAGTTTCCATACCTTCTGGTTCTGCCCAGCCTTCATAGTCCATTGGATTACC |
| 243 | CTTCTTTATCTTTATCTTCACTCAGACGATTACCTAGTACAGAACCTGCACTGCCTCCTCCAATGATGAC |
| 244 | CTTCTTTGCCTAATTCTCTACCGATACCTGATTGTTTGTATCCACCCCATGGCGCTTGTGCAAAATATGG |
| 245 | ATTGAATCGCTTCTTGTTCAGTTTCAAAGCCTTCTACAGTAACGACAGGTCCGAAAACCTCTTCTTGTAC |
| 246 | GATGCCTTGGCACTAGAAGCCGATGAAGGACGTTACTAACGACGATATGCTTTGGGGAGCTGTAAGTAAG |
| 247 | AGATTCATCAAGTGATTCCGACTCAGACAGTGATTCCGACTCAGACAGCGACTCCGAGTCAGGTTCTAAC |
| 248 | AGTGATTCCGACTCAGGTAGTGACTCCGATTCATCAAGTGATTCCGACTCAGACAGTGACTCAGACTCAG |
| 249 | TCGGATAGCGATTCAGATTCAGACAGCGATTCAGACTCAGATAATGACTCAGACTCAGACAGCGATTCGG |
| 250 | GCAGCTTGCTTACTTACTGCTGTACCTGTTATGAAAGTGTTCAAGTATTTTTATTCAAATCGCGGTCCAG |
| 251 | AGAGGTAGTTGATGGTGGTTTCCGCGTAAATGGTAAAGAAGTTAAATCATTCAGTGAACCAGATGCAAGC |
| 252 | ACAGGTGCTGCTAAAGCTATCGGTAAAGTTATTCCTGAAATCGATGGTAAATTAGATGGTGGTGCACAAC |
| 253 | AAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATCGCACATACAT |
| 254 | GATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCAT |
| 255 | CTAAGTAGCTCAGCAAATGCATCACAAACAGATAAYGGCGTAAATAGAAGTGGTTCTGAAGATCCAACAG |
| 256 | GTCGCTTTGCCCTTGGGTCATGCGTTGGTTCAATTCTTGGGCCAATCCTTCGGAAGATAGCATCTTTCCT |
| 257 | GGATGTATCGCCGAAGCAAGTTGTTTCAGCAGCGACAGCATGTATTCCATTCTTAGAAAATGATGACTCA |
The detection probes of table 8 C.jejuni (CJ), totally 17
| SEQ?ID?No. | 5 '------probe sequence------3 ' |
| 258 | ATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGCGC |
| 259 | GAAACCCTGACGCAGCAACGCCGCGTGGAGGATGACACTTTTCGGAGCGTAAACTCCTTTTCTTAGGGAA |
| 260 | GGTGCAAGCGTTACTCGGAATCACTGGGCGTAAAGGGCGCGTAGGCGGATTATCAAGTCTCTTGTGAAAT |
| 261 | AGTACGGTCGCAAGATTAAAACTCAAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA |
| 262 | TCATGGCCCTTATGCCCAGGGCGACACACGTGCTACAATGGCATATACAATGAGACGCAATACCGCGAGG |
| 263 | TAGCTTGCTAGAACTTAGAGACAGGTGCTGCACGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA |
| 264 | GGTCTTGTACTCACCGCCCGTCACACCATGGGAGTTGATTTCACTCGAAGCCGGAATACTAAACTAGTTA |
| 265 | GTAAGGAGTGCTGGAGCGAATAGAAGTGAGCATGCAGGCATGAGTAGCGATAATTAATGTGAGAATCATT |
| 266 | TGGCAAATTGGTTAATATTCCAATACCAACATTAGTGTGCGATGGAAGGACGCTTAGGGCTAAGGGGGCT |
| 267 | TAATGTTGCCCGTACCGTAAACCGACACAGGTGGGTGGGATGAGTATTCTAAGGCGCGTGGAAGAACTCT |
| 268 | AGCGGTGCTGATTTAGTACCTATTACTCCTATTACCCCACCTTTAACTAGAACAAGCAATAGTGCCAACA |
| 269 | CTAGCGGAGCAGCTTTAACGGTTTGGGCTTTAGCACAAGGAAATTGGATTTGGGGCTATACTTTAATCGA |
| 270 | GTCCATTATCCTTGYGATGCAAGCAATCACGCACAAATGTGGAAACTTATCCCTATGAGCAATACAGCGG |
| 271 | GTTGTAGGCGGGCTTACACCTATTCATCAATTTGTCAAAGTAGGTGAGGGTTGTATGATAGCAGGAGCAA |
| 272 | GAAGGGATTTTATTAAACACCTCTATTGCAAGCGTTGCTAGTGTTGCAGGGTTTAGTGTCCCAAGCTCTA |
| 273 | GGTAGAAATATCTTCGGTTATGTAACTGGTGGATATACTTTCAACGAAACAGTTCGCGTTGGTGCTGACT |
| 274 | TTGGTTTTAGATGCGCATGAGGGCTTTAACGAGCTTGATGAACGCATTGCTGGGCTTGTGGCTAAGCATT |
The detection probes of table 9 E.coli (EC), totally 73
| SEQ?ID?No. | 5 '------probe sequence---------3 ' |
| 275 | GAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGG |
| 276 | GATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTC |
| 277 | TGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGG |
| 278 | ACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGT |
| 279 | CTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCG |
| 280 | GAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGC |
| 281 | GGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGG |
| 282 | TGAATCAGTGTGTGTGTTAGTGGAAGCGTCTGGAAAGGCGCGCGATACAGGGTGACAGCCCCGTACACAA |
| 283 | GGAGAAGGCTATGTTGGCCGGGCGACGGTTGTCCCGGTTTAAGCGTGTAGGCTGGTTTTCCAGGCAAATC |
| 284 | GCAGTCAGAGGCGATGAAGGACGTGCTAATCTGCGATAAGCGTCGGTAAGGTGATATGAACCGTTATAAC |
| 285 | GGTCGCTGAATAAGCATTTATACAGTTCCGAAAGCGAAATGATGAAGGCTGCGCCTGGTCAGCAGATCAT |
| 286 | AAATGTCCCCGGACGTGACCAAAAGCAACATGACCGATGACAAGGCACTAAATTATGCGGCACAACAGGC |
| 287 | GTGCAAAAGTCAGCGAAGTTAATACAGAGGTTAAGGCTCCTGAGGTAAAATTCTTCTCAGTTCTGAGCAT |
| 288 | CCAAATTGTGAAAACGGATAAAGATGGTAGGGCTACGGTAAAACTGACATCTGGCGTTGCAGGTAATGCT |
| 289 | CAAGGCCAGCATTACTGAGATTAAGGCTGATAAAACAACAGCGAAGGCAAATGGTTCTGATGCGATTACC |
| 290 | CATGAAAATTAAAACTCTGGCAATCGTTGTTCTGTCGGCTCTGTCCCTCAGTTCTACAGCGGCTCTGGCC |
| 291 |
| AATGGTGGGACCGTTCACTTTAAAGGGGAAGTTGTTAACGCCGCTTGCGCAGTTGATGCAGGCTCTGTTG | |
| 292 | TTATTTTGCAACCGGTGCCGCAACCCCGGGTGCTGCTAATGCGGATGCGACCTTCAAGGTTCAGTATCAA |
| 293 | ATATCGATGGCGCGCAGAAAGCAACTGGCAGTGACCTGATTTCTAAATTTAAAGCGACAGGTACTGATAA |
| 294 | GCAATCTACCACCAACCCGCTCGAAACTATCGACAAAGCATTGGCTAAAGTTGACAATCTGCGTTCTGAC |
| 295 | CCGTAAACAACCTGTCTTCTGCCCGTAGCCGTATCGAAGATGCTGACTACGCGACCGAAGTGTCTAACAT |
| 296 | TATCAACAAGAACCAGTCTGCGCTGTCGAGTTCTATCGAGCGTCTGTCTTCTGGCTTGCGTATTAACAGC |
| 297 | GAAGCCAGGCATACGTAGACGATGCAGGCAACTTGACGACTAACAACGCTGGTAGCGCAGCTAAAGCTGA |
| 298 | GTAGTATTGAGCGAAACCAAAGCGGCTGCCGCGACATCTTCAATTACCTTTAATTCCGGTGTACTGAGCA |
| 299 | |
| 300 | CCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCTCAGTCTTCTCTGAGCTCCGCCATTGAACGTCTC |
| 301 | CCGTTTTACAGCAAATATTAAAGGTCTGACTCAGGCTTCCCGTAACGCGAATGATGGTATTTCTGTTGCG |
| 302 | GGACCAGAAGCTGTTAACGGATTTCCGCTCAGAACTACTCGATTCACGTTTTGGCGCAAAGGCCATTTCT |
| 303 | CTCGTCAGAACCTGGCCACTTTCTGCCAGACCTGGGACGACGAAAACGTCCATAAATTGATGGATTTGTC |
| 304 | GCAAGTAACGGATTTAAGGTCGGAACTACTCGATTCACGTTTTGGTGCGAAGTCTATTTCCACTATCGCA |
| 305 | ATTGGTTCTTCCGAGGCCTGTATGCTCGGCGGGATGGCGATGAAATGGCGTTGGCGCAAGCGTATGGAAG |
| 306 | TATGAGTTCATCTGTACGGGTCGCCCGGACGAAGGCATCCCGGCGGTTTGCTTCAAACTGAAAGATGGTG |
| 307 | GTCGCGGCTTCGAAATGGACTTTGCTGAACTGTTGCTGGAAGACTACAAAGCCTCCCTGAAATATCTCAG |
| 308 | ATCAACGGTTTTGGCCGTATCGGTCGCATTGTTTTCCGTGCTGCTCAGAAACGTTCTGACATCGAGATCG |
| 309 | ATCCGGCTAACCTGAAATGGGACGAAGTTGGTGTTGACGTTGTCGCTGAAGCAACTGGTCTGTTCCTGAC |
| 310 | TGATGACCACCGTTCACGCTACTACCGCTACTCAGAAAACCGTTGATGGCCCGTCTCACAAAGACTGGCG |
| 311 | GTAAAGTACTGCCAGAACTGAATGGCAAACTGACTGGTATGGCGTTCCGCGTTCCGACCCCGAACGTATC |
| 312 | AGATCAAAGCTGCCGTTAAAGCTGCTGCTGAAGGCGAAATGAAAGGCGTTCTGGGCTACACCGAAGATGA |
| 313 | ATCTACCGATTTCAACGGCGAAGTTTGCACTTCCGTGTTCGATGCTAAAGCTGGTATCGCTCTGAACGAC |
| 314 | TACAGAAATCTGAATATAGCTCCGGCAGAGGATGGTTACAGATTAGCAGGTTTCCCACCGGATCACCAAG |
| 315 | TAGCACCCGGTACAAGCAGGATTACAACACAATTCACAGCAGTAATTGCTACTATTCATGCTTTCAGGAC |
| 316 | GGTCTGAACGATATCCTCATCGCCTGTGTGGATGGCCTGAAAGGCTTCCCGGATGCCATCAACACAGTAT |
| 317 | CATCCAGTTATGCATCGTGCATATGGTGCGCAACAGCCTGCGCTTCGTGTCATGGAAGGACTACAAAGCC |
| 318 | CTTCCTGCTCGGTAAAACTAACGCCAGGAACCTGTGACAGCAGCGGCAGAATGGTAACACCAGAGTGACC |
| 319 | CCAATAACCGGCACTTCAACTTCGCCTGGCTGTTTGCCTTTCAGTTCCGCAACAAAGGTGTTGGAACGAA |
| 320 | TGTTAACCGGGTTAGTGATAATACCAATGCACGCTTTCGGGCAGGTTTTCGCAACTTGCTGTACCAGGTT |
| 321 | |
| 322 | AGGAACAGTATCCATGACATTCTCCGGTGTTACAGCAGGTGCAGGTAACGCCAATGACATGTTACAGACG |
| 323 | GGCAAACGTGTGAAGTTTGATGGTCGGACCTCTACCGGGGTTTCAAATCTGATTAATGGTGATAACACGA |
| 324 | CTTCTATTCACAGTGACAGGGTCATTCTGGCTATGAAGGACTATCTGGTAGGTGGGCACTCCCGTAAGGA |
| 325 | CTTCCTGAATACTGGGGAGAAGAACACGTATGGTGGGACGGCAGGGCTGCTTTTCATGGTGAGGTTGTCA |
| 326 | TGTGAATTTAACAGTCAGGGTGGGAACCTTTTCTCTGATTCCCGGATAAGGGTGACTTTCGATGGCGTCC |
| 327 | GGCGCTGTACGAGGTAAAGCCCGATGCCAGCATTCCTGACGACGATACGGAGCTGCTGCGCGATTACGTA |
| 328 | TGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCGGACACCAGAAATGCCTGTTCTG |
| 329 | ATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTT |
| 330 | CTCACCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATT |
| 331 | GGCATGACGTTATAGGCTACAATTATAGGATGACAAATATCTGCGCTGCTATAGGATTAGCCCAGTTAGA |
| 332 | ACTAGGACCGCAGAGGAAAGAGAGGAATTAAGGAATCACCTTGCAGATAAACTCATCGAAACAAGGCCAG |
| 333 | TTAGCTCAGTGGTGAGAGCGAGCGACTCATAATCGCCAGGTCGCTGGTTCAAATCCAGCAAGGGCCACCA |
| 334 | TTATATCTGCGCCGGGTCTGGTGCTGATTACTTCAGCCAAAAGGAACACCTGTATATGAAGTGTATATTA |
| 335 | CAGTTATTTTGCTGTGGATATACGAGGGCTTGATGTCTATCAGGCGCGTTTTGACCATCTTCGTCTGATT |
| 336 | ACATATATCAGTGCCCGGTGTGACAACGGTTTCCATGACAACGGACAGCAGTTATACCACTCTGCAACGT |
| 337 | ATTTCGTCAGGCACTGTCTGAAACTGCTCCTGTGTATACGATGACGCCGGGAGACGTGGACCTCACTCTG |
| 338 | TCCGGAGTATCGGGGAGAGGATGGTGTCAGAGTGGGGAGAATATCCTTTAATAATATATCAGCGATACTG |
| 339 | TACTGCAAAGTGCTCAGTTGACAGGAATGACTGTCACAATCAAATCCAGTACCTGTGAATCAGGCTCCGG |
| 340 | GCATAACCTGATTCGTGGTATGTGGGTAACAAGTGTAATCTGTGTCACAATTCAGTCAGTTGACAGTTGC |
| 341 | CCAGAATGGCATCTGATGAGTTTCCTTCTATGTGTCCGGCAGATGGAAGAGTCCGTGGGATTACGCACAA |
| 342 | TTTTCCAAGTATAATGAGGATGACACATTTACAGTGAAGGTTGACGGGAAAGAATACTGGACCAGTCGCT |
| 343 | AGAGTACTGGACTAACCGCTGGAATCTGCAACCGCTACTGCAAAGCGCACAGTTAACAGGAATGACGGTA |
| 344 | CAAAGTGTGGGTCAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTC |
| 345 | CGCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCGAACAGTTCCTGATYAACCACAAACCGTTCTACTTTA |
| 346 | TGGCAACCGGGTGAAGGTTATCTCTATGAACTGTGCGTCACAGCCAAAAGCCAGACAGAGTGTGATATCT |
| 347 | TACGTATCACHGTTTGTGTGAACAACGAACTGAACTGGCAGACTATCCCGCCGGGAATGGTGATTACCGA |
The positive and the negative control probe of table 10 Biosafood-8 chip, totally 4
| SEQ?ID?No. | 5 '------probe sequence------3 ' |
| 348 positive controls | GTTTGGCTGGTGTGGATCGTTTGGCTGGTGTGGATCGTTTGGCTGGTGTGGATCGTTTGGCTGGTGTGGA |
| 349 |
AAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAG |
| 350 negative control #2 | GCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAA |
| 351 |
ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTAC |
Three, the making of Biosafood-8 chip
1, the Biosafood-8 chip probe is synthetic
The Biosafood-8 probe groups has 351, wherein 47 of LM probe groups, 93 of VC probe groups, 33 of VP probe groups, 31 of Sal probe groups, 31 of Shi probe groups, 22 of SA probe groups, 17 of CJ probe groups, 73,1 positive control of EC probe groups and 3 negative controls amount to 351.(Integrated DNA Technologies Inc.) synthesizes to entrust American I DT.
" Oligo Specification Sheet " preparation 40 μ M probe stostes that provide after synthetic by IDT company; Change probe solution over to 384 orifice plates.
2, the design of Biosafood-8 chip array
The purpose of Biosafood-8 chip design is in order to detect 8 kinds of food-borne pathogens such as LM, VC, VP, Sal, Shi, SA, CJ and EC simultaneously, therefore, on this chip, will detect the probe of these 8 kinds of pathogenic bacterium, the detection probes with each bacterium makes up an inferior array respectively; And then 8 inferior Array Constructions that this is parallel are a big array.8 inferior arrays of the big array of this Biosafood-8 chip from top to bottom, are respectively that inferior array 1 is the LM probe groups; Inferior array 2 is the CV probe groups; Inferior array 3 is the VP probe groups; Inferior array 4 is the Sal probe groups; Inferior array 5 is the Shi probe groups; Inferior array 6 is the SA probe groups; Inferior array 7 is the CJ probe groups; Inferior array 8 is the EC probe groups.Each inferior array end is not enough to form the place of a policy in number of probes, all replaces with blank.For example last 7 array sites (L3:R34-54) of first inferior array LM array are blank spot.The end of last inferior array EC array that the positive control and the negative control probe of the big array of whole Biosafood-8 is placed on whole big array, be the positive contrast probe in L30:R34-45 position, L30:R46-48, L30:R49-51 and L30:R52-54 are respectively three negative probes.Between these 8 inferior arrays, blank with delegation respectively as the interval.
Each sample is printed 3 points, finally is printed as 18 * 3 * 30 array (Fig. 1).Each big array twice of duplicate printing on the chip slapper base.The layout of two big arrays on chip seen Fig. 2.
3, the printing of Biosafood-8 chip array
According to the operation instructions of chip point sample instrument, according to the design requirements of Biosafood-8 array, establishment chip point sample working procedure.Start the point sample program, the beginning point sample; After the program run of chip point sample finishes, take out 384 orifice plates,, go to the RT dark place and deposit with the envelope film phonograph seal.
After chip is printed, check chip print quality and effect with following two kinds of methods respectively.
Method 1: take out the chip that in high humidity environment, spends the night and handle, in microscope TE2000-E (Ecl ipse, Nikon) following form of observing probe points.Write down the various forms of chip probe point on the sheet base, comprised, diffusion, melting point, irregular, not of uniform size, array dislocation etc.
Method 2: with 6enePix 4100A chip scanner (Axon Instruments, Inc.) whole chip of scanning, sweep parameter be set to PMT gain=550; Resolving power is 10 μ m; Select the single length scanning of Cy3.With the scan image that obtains, deposit at last with " multilayer Tiff " form.
Four, the sample detection of gene chip
1, the cultivation of bacterium
From strain library, take out through traditional method and identify the bacterial strain of confirming, according to certain conditions recovery and separation and Culture separately; Collect an amount of lawn, be used for DNA extraction.
2. the extraction of genomic dna
Ordinary method is extracted the human gene group DNA.Adopt conventional pure manual extraction or centrifugal pillar test kit (worker company is given birth in Shanghai) to extract bacterial genomes DNA.Carry out the concentration of DNA and the mensuration of purity.
3. the mark of sample
The fluorochrome label method that is used for the sample of chip detection is used restricted universal joint (Restrictional General Adaptor) identified as samples notation [Li L, et al.A modified restrictiondisplay PCR method in sample-labelling of DNA microarray.J Virol Methods.2003; 114 (1): 71-75].The detected sample that is used for chip detection, after process Sau3AI enzyme is cut and is fractured into the short-movie section, these segmental two ends are connected with a universal joint with the otch coupling of this enzyme, use one and this universal joint paired universal primer then, pass through the amplification and the mark of the sample fragment of the restricted cutting of Sau3AI.With 5 ' end mark Cy3-UP (5 '〉Cy3-GTTTGGCTGGTGTGGATC＜3 ', Cy3-UP) as primer, it can carry out specificity with the RAF-RAR joint at the Sau3AI endonuclease bamhi two ends of sample DNA and combines, thereby with the mode mark sample dna fragment of pcr amplification.
Joint sequence is as follows:
RAF：5’>GATCCACACCAGCCAAACCCA<3’； MW：6378
RAR：5’>GGTTTGGCTGGTGTG<3’； MW：4775
4. the hybridization of chip
4.1 prehybridization
(1). the chip immersion is preheated to 55 ℃ prehybridization solution;
(2) place 150min for .55 ℃;
(3). clean totally 3 times with 0.1 SSC (elutriant III) under the room temperature, use IKA-WEAKE decolorization swinging table 150rpm jog 5 minutes at every turn;
(4). go to pure water, placed 30 seconds;
(5). with ChipMate (Tomy, PMC-082) centrifuge dripping chip; Place stand-by in cleaning place then.
4.2 hybridization
(1). in mark sample sediment tube, add 20L dH2O, 20 L2 * Hybridization Buffer (adding volume can adjust with the cover plate flexible in size), vibration mixing;
(2) .95 ℃ of insulation 10min; Use on the Minispin table whizzer at the centrifugal 2min of 13000rpm then;
(3) place the .RT dark place, prepares to be used to upload to chip array.
(4). get 40 μ L samples on the process array of prehybridization;
(5). on this array, cover the 12mm * 24mm cover glass of silanization processing;
(6). insert VersArrayTM chip hybridization box, in the aperture of hybridizing box two ends, respectively add 20 μ L water then, to keep its humidity;
(7). the hybridizing box that will be loaded with chip placed and is preheating to 55 ℃ water-bath, 55 ℃ of insulations 2～3 hours or spend the night.
4.3 clean behind the chip hybridization
(1). take out hybridizing box from 55 ℃ of water-baths, RT placed 3 minutes;
(2). from hybridizing box, take out chip gently, note not touching cover glass;
(3). rinse out hybridization solution and cover glass with tap water at ambient temperature;
(4). chip is gone among the elutriant I that is preheated to 55 ℃, in 55 ℃ of water-baths, place 10min;
(5). chip is gone among the elutriant II, use IKA-WEAKE decolorization swinging table 150rpm vibration 3min under the room temperature;
(6). in elutriant elutriant III, wash 3min;
(7). with ChipMate (Tomy, PMC-082) maximum speed of revolution (3500rpm) centrifuge dripping chip; Place chip storage box (Cat.No.:430052, Beijing Boao Biological Co., Ltd) then, place, wait detection to be scanned in clean dark place.
5. chip scanning
(1). and unlatching GenePix 4100A chip scanner (Axon Instruments, Inc.), more than the preheating 10min;
(2). open computer, operation GenePix Pro 5.1 (Axon Instruments, Inc.) chip scanning programs;
(3). click " Hardware Settings " icon, open operating parameter dialog box is set;
(4). the parameter of every setting is as follows:
Wavelength: 532nm; PMT gain=550; Filter:575DF35;
Pixel?Size：10μM；Lines?to?average：1；Focus?Position：0μM；
(5). with the sweep limit of " View Scan Area " command selection chip;
(6). clicking label is " Data Scan " icon in " Image " upper right corner, interface, the beginning chip scanning;
(7). in the chip scanning process, can use Loupe tool, magnified sweep zone, the details of observing scintigram;
(8). behind the end of scan, click the deposit icon,, deposit with " multilayer Tiff " form with the scan image that obtains.
6. the extraction of chip scanning data and analysis
(1). open computer, operation GenePix Pro 5.1;
(2). open the chip scanning picture of having stored;
(3). according to the dot matrix of array and inferior array definition extraction data;
(4). the size and the distance of the dot-matrix array of definition are carried out match with the chip dot matrix of actual scanning;
(5). adjust a left side, the right locating point that neutralizes, guarantee that all probe signals put complete match;
(6). select a plurality of inferior arrays, repeat above-mentioned match operation;
(7). click data extracts " Data Extraction " icon on right side, interface, and extraction data is converted to numeric data with view data;
(8). click save button as a result, store the data results that extracts;
(9). simultaneously, can be with the data of extracting, with the form derivation of " TXT " file;
(10). in preserving data options, select " Export Data As ", select the form of derived data to be " TXT " simultaneously;
(11). the TXT formatted data that derives is opened with " Excel " software;
(12). in Excel software, calculate the signal average of probe;
(13). all probe signals averages are gathered;
(14). the size according to the signal average is carried out data sorting;
(15). according to all positive probes in the positive probe signals value screening array of definition;
(16). calculate the PPR of each sample detection array;
(17). according to the PPR that the calculates gained figure that takes statistics;
(18). judge the ownership of sample according to result displayed in the statistical graph.
7. the sensitivity analysis of chip detection
Detected sample and sample mark joint are diluted downwards according to 10 times ratio, seek the absolute magnitude lower value of chip detection sample.
(1). get Listeria monocytogenes sample LM1 (06LM1407), its original concentration is 631.6 μ g/mL.At first, make its final concentration be about 30 μ g/mL (being 31.58 μ g/mL in fact) with 20 times of this diluted samples;
(2). get joint stoste (20 μ M) 10 μ L, add 10 μ L pure water, dilution becomes the concentration of 10 μ M, totally 20 μ L;
(3). according to the dilution scheme of following table (table 3-4), simultaneously sample solution and joint solution are carried out 10 times of dilution operations of series again.
In the experimental design, investigated 6 sample concentration Grad altogether, initial concentration is 30 μ g/mL, and minimum least concentration is 0.3ng/mL; Each sample size that adds is 3 μ L.
8. use the checking evaluation of Biosafood-8 chip detection known reference sample
With comprising that 8 food-borne pathogens detect the full array of Biosafood-8 chip of inferior array, detected the object bacteria of 8 kinds one by one, i.e. Listeria monocytogenes 3 strains, 3 strains of Ying Nuoke listeria bacteria, listeria ivanovii 1 strain, vibrio cholerae 3 strains, Vibrio parahaemolyticus 3 strains, Salmonellas 3 strains, Shigellae 3 strains, streptococcus aureus 3 strains, campylobacter jejuni 2 strains and escherichia coli 3 strains.Behind sample of Biosafood-8 chip detection, read the signal strength values of chip array probe and the hybridization of mark sample with chip scanner, with the format record deposit of chip scanning figure (TIFF).With GenePix Pro 5.1 software analysis and extract each chip probe and signal strength values that sample hybridization detects, be converted to numeric data then; Export to the Exel table with the form of TXT file and carry out positive number of probes quantitative analysis; For interpretation of result and the interpretation mode with a chip detection pathogenic bacterium sample, here we find by analyzing, and use after each chip array test sample the PPR on the array as parameter index, and are promptly directly perceived, efficient and convenient again.Behind sample of every detection, analyze 8 PPR behind this sample of inferior array detection respectively, and sort, determine the inferior array that PPR is the highest according to the height of PPR; At last, be that the result of test sample judges bacterial classification with the bacterial classification under the highest inferior array of PPR.Therefore, in this data analysis, all use the PPR of array to judge the specificity and the accuracy of a sample of chip detection.
8.1 the result of Biosafood-8 chip detection listeria sample
Listeria bacterium common in Guangdong Province's food has Listeria monocytogenes, Ying Nuoke listeria bacteria and Yi Shi (sheep) listeria bacteria.When three different listeria bacteria bacterial classification Listeria monocytogenes using Biosafood-8 chip detection listeria, Ying Nuoke listeria bacteria and listeria ivanovii, the PPR that the inferior array of LM shows is respectively 68.79%, 51.77%, 59.57%.Therefore, the Preliminary detection data presentation, these three different strains can make a distinction with the Biosafood-8 chip.
With Biosafood-8 chip detection Listeria monocytogenes (LM), 3 bacterial strain samples have been detected altogether.Fig. 3 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the Listeria monocytogenes of 04LM233; Fig. 4 has shown with behind three bacterial strains of this chip detection, carries out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three LM samples of Biosafood-8 chip detection, the PPR of the inferior array report of LM is the highest, and mean P PR is 68.79%, is no more than 25.5% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of LM is significantly higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.
8.2 the result of Biosafood-8 chip detection VC sample
With Biosafood-8 chip detection vibrio cholerae (VC), 3 bacterial strain samples have been detected altogether.Fig. 5 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the vibrio cholerae of V07-56;
After Fig. 6 has shown three bacterial strains of this chip detection, carry out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three VC samples of Biosafood-8 chip detection, the PPR of the inferior array report of VC is the highest, and mean P PR is 54.12%, is no more than 24.3% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of VC is significantly higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.
8.3 the result of Biosafood-8 chip detection VP sample
With Biosafood-8 chip detection Vibrio parahaemolyticus (VP), 3 bacterial strain samples have been detected altogether.Fig. 7 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the Vibrio parahaemolyticus of L-EP080; After Fig. 8 has shown three bacterial strains of this chip detection, carry out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three VP samples of Biosafood-8 chip detection, the PPR of the inferior array report of VP is the highest, and mean P PR is 66.67%, is no more than 19.7% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of VP is significantly higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.
8.4 the result of Biosafood-8 chip detection Sal sample
With Biosafood-8 chip detection Salmonellas (Sal), 3 bacterial strain samples have been detected altogether.Fig. 9 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the Salmonellas of 0705YS0167; After Figure 10 has shown three bacterial strains of this chip detection, carry out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three Sal samples of Biosafood-8 chip detection, the PPR of the inferior array report of Sal is the highest, and mean P PR is 55.91%, is no more than 21.6% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of Sal is significantly higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.
8.5 the result of Biosafood-8 chip detection Shi sample
With Biosafood-8 chip detection Shigellae (Shi), 3 bacterial strain samples have been detected altogether.Figure 11 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the Shigellae of L-SH166; After Figure 12 has shown three bacterial strains of this chip detection, carry out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three Shi samples of Biosafood-8 chip detection, the PPR of the inferior array report of Shi is the highest, and mean P PR is 53.76%, is no more than 50.6% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of Shi is apparently higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.It should be noted that, though the inferior array detection PPR of Sal is starkly lower than the positive rate of the inferior array of Shi, but when detecting the Shi sample, the former PPR seems higher, the specific findings that part probe in the inferior array of Sal is described is undesirable, when the next round optimization of this chip improves, also need constantly to increase the specific probe that detects the Shi sample.
8.6 the result of Biosafood-8 chip detection SA sample
With Biosafood-8 chip detection streptococcus aureus (SA), 3 bacterial strain samples have been detected altogether.Figure 13 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the streptococcus aureus of 05ST028; After Figure 14 has shown three bacterial strains of this chip detection, carry out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three SA samples of Biosafood-8 chip detection, the PPR of the inferior array report of SA is the highest, and mean P PR is 65.15%, is no more than 39.1% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of SA is significantly higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.But probe groups that it should be noted that the inferior array of LM is when detecting the SA sample, and its PPR is higher; To be gram-positive microorganism relevant with LM and SA probably for this, because with Biosafood-8 other gram-positive microorganism of chip detection such as swine streptococcus, streptococcus faecium and streptococcus pneumoniae the time, the inferior array of LM has also shown higher PPR.Even like this, because the PPR of the inferior array of SA is significantly higher than the positive rate of the inferior array of LM, so it does not influence the judgement of detected result.
8.7 the result of Biosafood-8 chip detection CJ/CC sample
Campylobacter is the gram negative bacilli of a class main parasitic in Mammals and bird enteron aisle.To the human disease mainly is the crooked bacterium of campylobacter jejuni and large intestine.These two generic bacterial classifications by the inferior array detection probe groups of the CJ of Biosafood-8 chip, can clearly clearly be identified and make a distinction.8.7.1 the result of Biosafood-8 chip detection CJ sample
With Biosafood-8 chip detection campylobacter jejuni (CJ), 3 bacterial strain samples have been detected altogether.Figure 15 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the campylobacter jejuni of 07C42; After Figure 16 has shown three bacterial strains of this chip detection, carry out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three CJ samples of Biosafood-8 chip detection, the PPR of the inferior array report of CJ is the highest, and mean P PR is 88.24%, is no more than 6.5% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of CJ is significantly higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.
8.7.2 the result of Biosafood-8 chip detection CC sample
With the Biosafood-8 chip detection crooked bacterium (CC) sample of 1 large intestine.Figure 17 has shown the chip scanning figure of this bacterium of Biosafood-8 chip detection; After Figure 18 has shown the crooked bacterium of this chip detection large intestine, carry out data analysis result relatively with detecting PPR.The result shows that behind the Biosafood-8 chip detection CC sample, the PPR of the inferior array report of CJ is the highest, and mean P PR is 58.82%, is no more than 9.7% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of CJ is significantly higher than the PPR of other inferior array; Because crooked bacterium of large intestine and campylobacter jejuni belong to same genus, homology is very high, so the actual test sample of Biosafood-8 chip detection result meets fully.But the PPR of the inferior array of CJ is 88.28% during Bisafood chip detection campylobacter jejuni, and the PPR of the inferior array of CJ is 58.28% when detecting the crooked bacterium of large intestine.Therefore, there were significant differences with the PPR of the crooked bacterium of two different strain campylobacter jejunis of this chip detection generic and large intestine, can clearly two bacterial classifications be made a distinction fully.
8.8 the result of Biosafood-8 chip detection EC sample
With Biosafood-8 chip detection escherichia coli (EC), 3 bacterial strain samples have been detected altogether.Figure 19 has shown that Biosafood-8 chip detection strain number is the chip scanning figure of the escherichia coli of EC882364; After Figure 20 has shown three bacterial strains of this chip detection, carry out data analysis result relatively with the PPR average that three bacterial strains detect.The result shows that behind three EC samples of Biosafood-8 chip detection, the PPR of the inferior array report of EC is the highest, and mean P PR is 47.95%, is no more than 36.6% and the mean P PR of other 7 the inferior arrays of detection is the highest; The PPR of the inferior array of EC is significantly higher than the PPR of other inferior array; Therefore the actual test sample of Biosafood-8 chip detection result meets fully.
Sum up the detected result of above-mentioned 8 the food-borne pathogens kinds of Biosafood-8 chip detection, find that this chip can be by 8 integrated object bacteria detection probes Asia arrays, special and show detected object bacteria sample accurately.When detecting the object bacteria sample, hybridize earlier detected result per sample, simultaneously the PPR of 8 inferior arrays on the analysis chip; Then, the PPR according to each inferior array just can determine the inferior array that PPR is the highest; Just can judge at last that the bacterial classification of that inferior array correspondence that PPR is the highest is exactly the bacterial classification under the detected sample.In theory, 8 food-borne pathogens of on the Biosafood-8 chip this detect inferior array, when detecting the target bacterial classification, PPR should be 100%, for example, on the inferior array of VC, integrated all is the detection probes of particular target gene on the VC genome, therefore when detecting the VC sample, the VC probe should find the target dna fragment to coupling.But in actual detected,, therefore, on the inferior array of VC, will there be the false negative probe (see for details part is discussed) of some amount because the defective of sample marking method can not realize allowing the full genomic fragment of VC sample all be labeled.The situation of other inferior array is also similar on this chip.This Biosafood-8 chip is when detecting inferior array enantiomorphous target sample, and actual PPR is respectively: the PPR of the inferior array detection LM of LM sample is 68.79%, and detecting generic Lin is 51.77%, and Liv is 59.57%; The inferior array detection VC of VC sample 54.12%; The inferior array detection VP of VP sample 66.67%; The inferior array detection Sal of Sal sample 55.91%; The inferior array detection Shi of Shi sample 53.76; The inferior array detection SA of SA sample 65.15%; The inferior array detection CJ of CJ sample 88.24% detects generic CC sample 58.82%; The inferior array detection EC of EC sample 47.95%.Top these group data are arranged as can be seen, 8 food-borne pathogens on the Biosafood-8 chip detect the PPR of inferior array detection target bacterial classification, at each inferior array, it is a more stable value, but at different inferior arrays, variation range strengthens, and minimum is the inferior array 47.95% of escherichia coli, and the highest is campylobacter jejuni array 88.24%; And the PPR of all these 8 inferior arrays is not less than 47%.Therefore can be set at a threshold value to 45% this value a little less than the above-mentioned minimum inferior array PPR of EC with Biosafood-8 chip detection food-borne pathogens object bacteria and non-object bacteria.That is to say, when with the suspicious food poisoning bacterium sample of Biosafood-8 chip detection the unknown, the PPR of 8 inferior array detection result's reports on chip, if the inferior array of none surpasses 45%, so, just can think that this detected sample is other bacteria samples outside these 8 kinds.This is a preliminary hypothesis according to a judgement sample of above-mentioned detection data proposition.Whether this hypothesis is set up, and also needs sample more with the Biosafood-8 chip detection, greater amt, verifies this hypothesis.
Detect assessment with some non-object bacteria samples below, verify the part work of this hypothesis exactly.
9, use the result of other non-object bacteria sample of Biosafood-8 chip detection
Specificity and reliability for thoroughly evaluating Biosafood-8 chip detection, the contriver has tentatively detected except that above-mentioned 8 bacterium other with the Biosafood-8 chip especially certain representational biological sample, as non-object bacteria samples such as Lambda phage, people's gene group, streptococcus pneumoniae, swine streptococcus, streptococcus faecium, Pseudomonas aeruginosa and Neisseria meningitidiss; Found that, in these detected samples, except three of the streptococcus blue look positive bacterias of leather the PPR of the inferior array of LM is higher, all the other samples of Biosafood-8 chip detection have all shown good detection specificity.
With the Biosafood-8 chip detection 1 Lambda phage sample.Figure 21 has shown the chip scanning figure of this sample of Biosafood-8 chip detection; Figure 22 carries out data analysis result relatively with detecting PPR after having shown this chip detection virus Lambda phage DNA.The result shows, behind the Biosafood-8 chip detection Lambda phage sample, the PPR of the inferior array that detects at 8 food-borne pathogens is generally very low, two inferior arrays of VC and Sal indivedual probe positives only wherein, but PPR not have above 3.3%; The PPR of all the other inferior arrays is 0.00%.Therefore the non-specific hybridization signal of Biosafood-8 chip detection Lambda phage is extremely low.
With the Biosafood-8 chip detection 1 people's complete genome DNA sample.Figure 23 has shown the chip scanning figure of this sample of Biosafood-8 chip detection; After Figure 24 has shown this chip detection people complete genome DNA, carry out data analysis result relatively with detecting PPR.The result shows, behind the Biosafood-8 chip detection people complete genome DNA sample, the PPR of the inferior array that detects at 8 food-borne pathogens is generally very low, VC, Sal, Shi and four inferior arrays of EC indivedual probe positives only wherein, but PPR not have above 9.7%; The PPR of all the other inferior arrays is 0.00%.Therefore the non-specific hybridization signal of Biosafood-8 chip detection human gene group DNA sample is extremely low.
With the Biosafood-8 chip detection 1 streptococcus pneumoniae sample.Figure 25 has shown the chip scanning figure of this bacterium of Biosafood-8 chip detection; After Figure 26 has shown this chip detection streptococcus pneumoniae, carry out data analysis result relatively with detecting PPR.The result shows, behind the Biosafood-8 chip detection streptococcus pneumoniae sample, the PPR of the inferior array that detects at 8 food-borne pathogens is generally lower, but wherein has the PPR of LM, SA and three inferior arrays of CJ higher, but PPR not have above 34.1%; The PPR of all the other inferior arrays is very low, does not surpass 9.7%.Therefore the non-specific hybridization signal of Biosafood-8 chip detection streptococcus pneumoniae sample is lower.
With the Biosafood-8 chip detection 1 swine streptococcus sample.Figure 27 has shown the chip scanning figure of this bacterium of Biosafood-8 chip detection; After Figure 28 has shown this chip inspection swine streptococcus, carry out data analysis result relatively with detecting PPR.The result shows, behind the Biosafood-8 chip detection swine streptococcus sample, the PPR of the inferior array that detects at 8 food-borne pathogens is generally lower, but wherein has the PPR of LM and two inferior arrays of CJ higher, but PPR not have above 42.6%; The PPR of all the other inferior arrays is very low, does not surpass 29.1%.Therefore the non-specific hybridization signal of Biosafood-8 chip detection swine streptococcus sample is lower.It should be noted that the inferior array detection swine streptococcus of LM sample is to have shown higher PPR 42.55%.On this explanation swine streptococcus genome with the LM probe groups of Biosafood-8 chip in probe have the sequence of high homology more, when test set suis sample, give special heed to.But according to the data of front as can be seen, therefore the PPR of the inferior array detection target sample LM of LM and generic Lin or Liv still can make a distinction them clearly more than 51%.
With the Biosafood-8 chip detection 1 streptococcus faecium sample.Figure 29 has shown the chip scanning figure of this bacterium of Biosafood-8 chip detection; After Figure 30 has shown this chip detection streptococcus faecium, carry out data analysis result relatively with detecting PPR.The result shows, behind the Biosafood-8 chip detection streptococcus faecium sample, the PPR of the inferior array that detects at 8 food-borne pathogens is generally lower, but wherein the PPR of the inferior array of LM is higher, but PPR not have above 36.2%; The PPR of all the other inferior arrays is very low, does not surpass 23.6%.Therefore the non-specific hybridization signal of Biosafood-8 chip detection streptococcus faecium sample is lower.
With the Biosafood-8 chip detection 1 Neisseria meningitidis sample.Figure 31 has shown the chip scanning figure of this bacterium of Biosafood-8 chip detection; After Figure 32 has shown this chip detection Neisseria meningitidis, carry out data analysis result relatively with detecting PPR.The result shows, behind the Biosafood-8 chip detection Neisseria meningitidis sample, the PPR of the inferior array that detects at 8 food-borne pathogens is generally lower, but wherein the PPR of the inferior array of CJ is higher, but PPR not have above 29.5%; The PPR of all the other inferior arrays is very low, does not surpass 19.4%.Therefore the non-specific hybridization signal of Biosafood-8 chip detection Neisseria meningitidis sample is lower.
With the Biosafood-8 chip detection 1 Pseudomonas aeruginosa sample.Figure 33 has shown the chip scanning figure of this bacterium of Biosafood-8 chip detection; After Figure 34 has shown this chip detection Pseudomonas aeruginosa, carry out data analysis result relatively with detecting PPR.The result shows, behind the Biosafood-8 chip detection Pseudomonas aeruginosa sample, the PPR of the inferior array that detects at 8 food-borne pathogens is generally lower, but wherein the PPR of Sal and two inferior arrays of CJ is higher, but PPR not have above 29.5%; The PPR of all the other inferior arrays is very low, does not surpass 19.4%.Therefore the non-specific hybridization signal of Biosafood-8 chip detection Pseudomonas aeruginosa sample is lower.
Analysis and summary show that with other sample outside 8 object bacteria of Biosafood-8 chip detection, result these non-object bacteria samples are used the Biosafood-8 chip detection, have shown that all lower or very low-level hybridization is non-specific.This group detects assessment data, in conjunction with the detection data of the target food-borne pathogens of 8 kinds of front Biosafood-8 chip detection, has confirmed the exactness of this Biosafood-8 chip detection sample fully.Simultaneously, the hypothesis with the inferior array detection PPR 45% conduct differentiation tentative pathogenic bacterium of target of Biosafood-8 chip and other non-specific sample critical value that proposes previously by the detection validation of this part sample of back, remains correct.With these non-object bacteria samples of Biosafood-8 chip detection, what inferior array PPR was minimum is 0.00%, and the highest be 42.55% of the inferior array detection swine streptococcus of LM sample.By the separating capacity of the inferior array detection all samples of analysis-by-synthesis LM, as can be seen, the specificity of the inferior array detection target sample of LM is significantly higher than non-object bacteria sample.
Therefore, complete detection analysis by above-mentioned 18 kinds of known reference kind samples, tentative confirmation, the Biosafood-8 food-borne pathogens detection chip of the present invention's development, can know correctly to detect 8 kinds of target food-borne pathogens, also can get rid of 8 target food-borne pathogens kinds other non-object bacteria sample in addition clearly.
Above-mentioned checking assessment to the Biosafood-8 chip is under the framework of the inferior array of 8 object bacteria of chip piece, and once assessment detects specificity and the exactness that entire chip detects a sample; In other words conj.or perhaps with " mode or the angle of a sample of 8 inferior arrays ", the detection performance of whole Biosafood-8 chip.This also is the mode with the practical application of Biosafood-8 chip, simulates Biosafood-8 in other words and carries out the checking assessment that mode that actual sample detects is carried out.In order further to assess the detection performance of Biosafood-8 chip, make full use of Biosafood-8 and detect the information that data provided that obtains behind the sample of 18 kinds, further from " angles of 18 samples of an inferior array ", investigate the detection performance of analyzing the Biosafood-8 chip below.
10, to the specific checking assessment of each inferior array detection on the Biosafood-8 chip
With 18 Biosafood-8 chip detection behind the sample of 18 different generas, in fact obtain all to detect data, can be divided into two groups of data.First group of data is that from the angle of sample, each detected sample all passes through the detection of 8 inferior arrays on the chip piece, thereby obtains to detect simultaneously at 8 inferior arrays 8 PPR data of this sample.By analyzing this group data, can determine the specificity of whole each sample of Biosafood-8 chip detection; Above-mentioned comparative analysis is finished at last branch.Second group of data be, the angle of each inferior array from the Biosafood-8 chip, and each inferior array all detects 18 samples, has obtained 18 PPR data.
The result data of the sample by extracting 18 different generas sources of reductive analysis Biosafood-8 chip detection as the object of investigating, has been analyzed the sample detection performance of single inferior array on this chip with each inferior array on the Biosafood-8 chip.The result shows, carries out the ordering of PPR behind 18 kind samples of this actual detected and confirms that relatively the PPR that each target food-borne pathogens detects inferior array detection respective objects bacterium is the highest, actual detected result and desired value (Figure 35) in full accord.All the other samples though also demonstrate in various degree PPR, generally are starkly lower than the PPR of target sample.The preliminary identification of the known reference sample by these 18 kinds verifies, and 8 food-borne pathogens on the Biosafood-8 chip detect inferior array can distinguish target pathogenic bacterium sample and non-target pathogenic bacterium sample clear and clearly.
SEQUENCE LISTING (sequence table)
＜110〉Guangdong Prov. Disease Prevention-control Center
＜120〉composite gene chip of food-borne pathogens detection
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<400>130
<210>131
<211>70
<212>DNA
＜213〉artificial sequence
<400>131
<210>132
<211>70
<212>DNA
＜213〉artificial sequence
<400>132
<210>133
<211>70
<212>DNA
＜213〉artificial sequence
<400>133
<210>134
<211>70
<212>DNA
＜213〉artificial sequence
<400>134
<210>135
<211>70
<212>DNA
＜213〉artificial sequence
<400>135
<210>136
<211>70
<212>DNA
＜213〉artificial sequence
<400>136
<210>137
<211>70
<212>DNA
＜213〉artificial sequence
<400>137
<210>138
<211>70
<212>DNA
＜213〉artificial sequence
<400>138
<210>139
<211>70
<212>DNA
＜213〉artificial sequence
<400>139
<210>140
<211>70
<212>DNA
＜213〉artificial sequence
<400>140
<210>141
<211>70
<212>DNA
＜213〉artificial sequence
<400>141
<210>142
<211>70
<212>DNA
＜213〉artificial sequence
<400>142
<210>143
<211>70
<212>DNA
＜213〉artificial sequence
<400>143
<210>144
<211>70
<212>DNA
＜213〉artificial sequence
<400>144
<210>145
<211>70
<212>DNA
＜213〉artificial sequence
<400>145
<210>146
<211>70
<212>DNA
＜213〉artificial sequence
<400>146
<210>147
<211>70
<212>DNA
＜213〉artificial sequence
<400>147
<210>148
<211>70
<212>DNA
＜213〉artificial sequence
<400>148
<210>149
<211>70
<212>DNA
＜213〉artificial sequence
<400>149
<210>150
<211>70
<212>DNA
＜213〉artificial sequence
<400>150
<210>151
<211>70
<212>DNA
＜213〉artificial sequence
<400>151
<210>152
<211>70
<212>DNA
＜213〉artificial sequence
<400>152
<210>153
<211>70
<212>DNA
＜213〉artificial sequence
<400>153
<210>154
<211>70
<212>DNA
＜213〉artificial sequence
<400>154
<210>155
<211>70
<212>DNA
＜213〉artificial sequence
<400>155
<210>156
<211>70
<212>DNA
＜213〉artificial sequence
<400>156
<210>157
<211>70
<212>DNA
＜213〉artificial sequence
<400>157
<210>158
<211>70
<212>DNA
＜213〉artificial sequence
<400>158
<210>159
<211>70
<212>DNA
＜213〉artificial sequence
<400>159
<210>160
<211>70
<212>DNA
＜213〉artificial sequence
<400>160
<210>161
<211>70
<212>DNA
＜213〉artificial sequence
<400>161
<210>162
<211>70
<212>DNA
＜213〉artificial sequence
<400>162
<210>163
<211>70
<212>DNA
＜213〉artificial sequence
<400>163
<210>164
<211>70
<212>DNA
＜213〉artificial sequence
<400>164
<210>165
<211>70
<212>DNA
＜213〉artificial sequence
<400>165
<210>166
<211>70
<212>DNA
＜213〉artificial sequence
<400>166
<210>167
<211>70
<212>DNA
＜213〉artificial sequence
<400>167
<210>168
<211>70
<212>DNA
＜213〉artificial sequence
<400>168
<210>169
<211>70
<212>DNA
＜213〉artificial sequence
<400>169
<210>170
<211>70
<212>DNA
＜213〉artificial sequence
<400>170
<210>171
<211>70
<212>DNA
＜213〉artificial sequence
<400>171
<210>172
<211>70
<212>DNA
＜213〉people's operation
<400>172
<210>173
<211>70
<212>DNA
＜213〉artificial sequence
<400>173
<210>174
<211>70
<212>DNA
＜213〉artificial sequence
<400>174
<210>175
<211>70
<212>DNA
＜213〉artificial sequence
<400>175
<210>176
<211>70
<212>DNA
＜213〉artificial sequence
<400>176
<210>177
<211>70
<212>DNA
＜213〉artificial sequence
<400>177
<210>178
<211>70
<212>DNA
＜213〉artificial sequence
<400>178
<210>179
<211>70
<212>DNA
＜213〉artificial sequence
<400>179
<210>180
<211>70
<212>DNA
＜213〉artificial sequence
<400>180
<210>181
<211>70
<212>DNA
＜213〉artificial sequence
<400>181
<210>182
<211>70
<212>DNA
＜213〉artificial sequence
<400>182
<210>183
<211>70
<212>DNA
＜213〉artificial sequence
<400>183
<210>184
<211>70
<212>DNA
＜213〉artificial sequence
<400>184
<210>185
<212>DNA
＜213〉artificial sequence
<400>185
<210>186
<211>70
<212>DNA
＜213〉artificial sequence
<400>186
<210>187
<211>70
<212>DNA
＜213〉artificial sequence
<400>187
<210>188
<211>70
<212>DNA
＜213〉artificial sequence
<400>188
<210>189
<211>70
<212>DNA
＜213〉artificial sequence
<400>189
<210>190
<211>70
<212>DNA
＜213〉artificial sequence
<400>190
<210>191
<211>70
<212>DNA
＜213〉artificial sequence
<400>191
<210>192
<211>70
<212>DNA
＜213〉artificial sequence
<400>192
<210>193
<211>70
<212>DNA
＜213〉artificial sequence
<400>193
<210>194
<211>70
<212>DNA
＜213〉artificial sequence
<400>194
<210>195
<211>70
<212>DNA
＜213〉artificial sequence
<400>195
<210>196
<211>70
<212>DNA
＜213〉artificial sequence
<400>196
<210>197
<211>70
<212>DNA
＜213〉artificial sequence
<400>197
<210>198
<211>70
<212>DNA
＜213〉artificial sequence
<400>198
<210>199
<211>70
<212>DNA
＜213〉artificial sequence
<400>199
<210>200
<211>70
<212>DNA
＜213〉artificial sequence
<400>200
<210>201
<211>70
<212>DNA
＜213〉artificial sequence
<400>201
<210>202
<211>70
<212>DNA
＜213〉artificial sequence
<400>202
<210>203
<211>70
<212>DNA
＜213〉artificial sequence
<400>203
<210>204
<211>70
<212>DNA
＜213〉artificial sequence
<400>204
<210>205
<211>70
<212>DNA
＜213〉artificial sequence
<400>205
<210>206
<211>70
<212>DNA
＜213〉artificial sequence
<400>206
<210>207
<211>70
<212>DNA
＜213〉artificial sequence
<400>207
<210>208
<211>70
<212>DNA
＜213〉artificial sequence
<400>208
<210>209
<211>70
<212>DNA
＜213〉artificial sequence
<400>209
<210>210
<211>70
<212>DNA
＜213〉artificial sequence
<400>210
<210>211
<211>70
<212>DNA
＜213〉artificial sequence
<400>211
<210>212
<211>70
<212>DNA
＜213〉artificial sequence
<400>212
<210>213
<211>70
<212>DNA
＜213〉artificial sequence
<400>213
<210>214
<211>70
<212>DNA
＜213〉artificial sequence
<400>214
<210>215
<211>70
<212>DNA
＜213〉artificial sequence
<400>215
<210>216
<211>70
<212>DNA
＜213〉artificial sequence
<400>216
<210>217
<211>70
<212>DNA
＜213〉artificial sequence
<400>217
<210>218
<211>70
<212>DNA
＜213〉artificial sequence
<400>218
<210>219
<211>70
<212>DNA
＜213〉artificial sequence
<400>219
<210>220
<211>70
<212>DNA
＜213〉artificial sequence
<400>220
<210>221
<211>70
<212>DNA
＜213〉artificial sequence
<400>221
<210>222
<211>70
<212>DNA
＜213〉artificial sequence
<400>222
<210>223
<211>70
<212>DNA
＜213〉artificial sequence
<400>223
<210>224
<211>70
<212>DNA
＜213〉artificial sequence
<400>224
<210>225
<211>70
<212>DNA
＜213〉artificial sequence
<400>225
<210>226
<211>70
<212>DNA
＜213〉artificial sequence
<400>226
<210>227
<211>70
<212>DNA
＜213〉artificial sequence
<400>227
<210>228
<211>70
<212>DNA
＜213〉artificial sequence
<400>228
<210>229
<211>70
<212>DNA
＜213〉artificial sequence
<400>229
<210>230
<211>70
<212>DNA
＜213〉artificial sequence
<400>230
<210>231
<211>70
<212>DNA
＜213〉artificial sequence
<400>231
<210>232
<211>70
<212>DNA
＜213〉artificial sequence
<400>232
<210>233
<211>70
<212>DNA
＜213〉artificial sequence
<400>233
<210>234
<211>70
<212>DNA
＜213〉artificial sequence
<400>234
<210>235
<211>70
<212>DNA
＜213〉artificial sequence
<400>235
<210>236
<211>70
<212>DNA
＜213〉artificial sequence
<400>236
<210>237
<211>70
<212>DNA
＜213〉artificial sequence
<400>237
<210>238
<211>70
<212>DNA
＜213〉artificial sequence
<400>238
<210>239
<211>70
<212>DNA
＜213〉artificial sequence
<400>239
<210>240
<211>70
<212>DNA
＜213〉artificial sequence
<400>240
<210>241
<211>70
<212>DNA
＜213〉artificial sequence
<400>241
<210>242
<211>70
<212>DNA
＜213〉artificial sequence
<400>242
<210>243
<211>70
<212>DNA
＜213〉artificial sequence
<400>243
<210>244
<211>70
<212>DNA
＜213〉artificial sequence
<400>244
<210>245
<211>70
<212>DNA
＜213〉artificial sequence
<400>245
<210>246
<211>70
<212>DNA
＜213〉artificial sequence
<400>246
<210>247
<211>70
<212>DNA
＜213〉artificial sequence
<400>247
<210>248
<211>70
<212>DNA
＜213〉artificial sequence
<400>248
<210>249
<211>70
<212>DNA
＜213〉artificial sequence
<400>249
<210>250
<211>70
<212>DNA
＜213〉artificial sequence
<400>250
<210>251
<211>70
<212>DNA
＜213〉artificial sequence
<400>251
<210>252
<211>70
<212>DNA
＜213〉artificial sequence
<400>252
<210>253
<211>70
<212>DNA
＜213〉artificial sequence
<400>253
<210>254
<211>70
<212>DNA
＜213〉artificial sequence
<400>254
<210>255
<211>70
<212>DNA
＜213〉artificial sequence
<400>255
<210>256
<211>70
<212>DNA
＜213〉artificial sequence
<400>256
<210>257
<211>70
<212>DNA
＜213〉artificial sequence
<400>257
<210>258
<211>70
<212>DNA
＜213〉artificial sequence
<400>258
<210>259
<211>70
<212>DNA
＜213〉artificial sequence
<400>259
<210>260
<211>70
<212>DNA
＜213〉artificial sequence
<400>260
<210>261
<211>70
<212>DNA
＜213〉artificial sequence
<400>261
<210>262
<211>70
<212>DNA
＜213〉artificial sequence
<400>262
<210>263
<211>70
<212>DNA
＜213〉artificial sequence
<400>263
<210>264
<211>70
<212>DNA
＜213〉artificial sequence
<400>264
<210>265
<211>70
<212>DNA
＜213〉artificial sequence
<400>265
<210>266
<211>70
<212>DNA
＜213〉artificial sequence
<400>266
<210>267
<211>70
<212>DNA
＜213〉artificial sequence
<400>267
<210>268
<211>70
<212>DNA
＜213〉artificial sequence
<400>268
<210>269
<211>70
<212>DNA
＜213〉artificial sequence
<400>269
<210>270
<211>70
<212>DNA
＜213〉artificial sequence
<400>270
<210>271
<211>70
<212>DNA
＜213〉artificial sequence
<400>271
<210>272
<211>70
<212>DNA
＜213〉artificial sequence
<400>272
<210>273
<211>70
<212>DNA
＜213〉artificial sequence
<400>273
<210>274
<211>70
<212>DNA
＜213〉artificial sequence
<400>274
<210>275
<211>70
<212>DNA
＜213〉artificial sequence
<400>275
<210>276
<211>70
<212>DNA
＜213〉artificial sequence
<400>276
<210>277
<211>70
<212>DNA
＜213〉artificial sequence
<400>277
<210>278
<211>70
<212>DNA
＜213〉artificial sequence
<400>278
<210>279
<211>70
<212>DNA
＜213〉artificial sequence
<400>279
<210>280
<211>70
<212>DNA
＜213〉artificial sequence
<400>280
<210>281
<211>70
<212>DNA
＜213〉artificial sequence
<400>281
<210>282
<211>70
<212>DNA
＜213〉artificial sequence
<400>282
<210>283
<211>70
<212>DNA
＜213〉artificial sequence
<400>283
<210>284
<211>70
<212>DNA
＜213〉artificial sequence
<400>284
<210>285
<211>70
<212>DNA
＜213〉artificial sequence
<400>285
<210>286
<211>70
<212>DNA
＜213〉artificial sequence
<400>286
<210>287
<211>70
<212>DNA
＜213〉artificial sequence
<400>287
<210>288
<211>70
<212>DNA
＜213〉artificial sequence
<400>288
<210>289
<211>70
<212>DNA
＜213〉artificial sequence
<400>289
<210>290
<211>70
<212>DNA
＜213〉artificial sequence
<400>290
<210>291
<211>70
<212>DNA
＜213〉artificial sequence
<400>291
<210>292
<211>70
<212>DNA
＜213〉artificial sequence
<400>292
<211>70
<212>DNA
＜213〉artificial sequence
<400>293
<210>294
<211>70
<212>DNA
＜213〉artificial sequence
<400>294
<210>295
<211>70
<212>DNA
＜213〉artificial sequence
<400>295
<210>296
<211>70
<212>DNA
＜213〉artificial sequence
<400>296
<210>297
<211>70
<212>DNA
＜213〉artificial sequence
<400>297
<210>298
<211>70
<212>DNA
＜213〉artificial sequence
<400>298
<210>299
<211>70
<212>DNA
＜213〉artificial sequence
<400>299
<210>300
<211>70
<212>DNA
＜213〉artificial sequence
<400>300
<210>301
<211>70
<212>DNA
＜213〉artificial sequence
<400>301
<210>302
<211>70
<212>DNA
＜213〉artificial sequence
<400>302
<210>303
<211>70
<212>DNA
＜213〉artificial sequence
<400>303
<210>304
<211>70
<212>DNA
＜213〉artificial sequence
<400>304
<210>305
<211>70
<212>DNA
＜213〉artificial sequence
<400>305
<210>306
<211>70
<212>DNA
＜213〉artificial sequence
<400>306
<210>307
<211>70
<212>DNA
＜213〉artificial sequence
<400>307
<210>308
<211>70
<212>DNA
＜213〉artificial sequence
<400>308
<210>309
<211>70
<212>DNA
＜213〉artificial sequence
<400>309
<210>310
<211>70
<212>DNA
＜213〉artificial sequence
<400>310
<210>311
<211>70
<212>DNA
＜213〉artificial sequence
<400>311
<210>312
<211>70
<212>DNA
＜213〉artificial sequence
<400>312
<210>313
<211>70
<212>DNA
＜213〉artificial sequence
<400>313
<210>314
<211>70
<212>DNA
＜213〉artificial sequence
<400>314
<210>315
<211>70
<212>DNA
＜213〉artificial sequence
<400>315
<210>316
<211>70
<212>DNA
＜213〉artificial sequence
<400>316
<210>317
<211>70
<212>DNA
＜213〉artificial sequence
<400>317
<210>318
<211>70
<212>DNA
＜213〉artificial sequence
<400>318
<210>319
<211>70
<212>DNA
＜213〉artificial sequence
<400>319
<210>320
<211>70
<212>DNA
＜213〉artificial sequence
<400>320
<210>321
<211>70
<212>DNA
＜213〉artificial sequence
<400>321
<210>322
<211>70
<212>DNA
＜213〉artificial sequence
<400>322
<210>323
<211>70
<212>DNA
＜213〉artificial sequence
<400>323
<210>324
<211>70
<212>DNA
＜213〉artificial sequence
<400>324
<210>325
<211>70
<212>DNA
＜213〉artificial sequence
<400>325
<210>326
<211>70
<212>DNA
＜213〉artificial sequence
<400>326
<210>327
<211>70
<212>DNA
＜213〉artificial sequence
<400>327
<210>328
<211>70
<212>DNA
＜213〉artificial sequence
<400>328
<210>329
<211>70
<212>DNA
＜213〉artificial sequence
<400>329
<210>330
<211>70
<212>DNA
＜213〉artificial sequence
<400>330
<210>331
<211>70
<212>DNA
＜213〉artificial sequence
<400>331
<210>332
<211>70
<212>DNA
＜213〉artificial sequence
<400>332
<210>333
<211>70
<212>DNA
＜213〉artificial sequence
<400>333
<210>334
<211>70
<212>DNA
＜213〉artificial sequence
<400>334
<210>335
<211>70
<212>DNA
＜213〉artificial sequence
<400>335
<210>336
<211>70
<212>DNA
＜213〉artificial sequence
<400>336
<210>337
<211>70
<212>DNA
＜213〉artificial sequence
<400>337
<210>338
<211>70
<212>DNA
＜213〉artificial sequence
<400>338
<210>339
<211>70
<212>DNA
＜213〉artificial sequence
<400>339
<210>340
<211>70
<212>DNA
＜213〉artificial sequence
<400>340
<210>341
<211>70
<212>DNA
＜213〉artificial sequence
<400>341
<210>342
<211>70
<212>DNA
＜213〉artificial sequence
<400>342
<210>343
<211>70
<212>DNA
＜213〉artificial sequence
<400>343
<210>344
<211>70
<212>DNA
＜213〉artificial sequence
<400>344
<210>345
<211>70
<212>DNA
＜213〉artificial sequence
<400>345
<210>346
<211>70
<212>DNA
＜213〉artificial sequence
<400>346
<210>347
<211>70
<212>DNA
＜213〉artificial sequence
<400>347
<210>348
<211>70
<212>DNA
＜213〉artificial sequence
<400>348
<210>349
<211>70
<212>DNA
＜213〉artificial sequence
<400>349
<210>350
<211>70
<212>DNA
＜213〉artificial sequence
<400>350
<210>351
<211>70
<212>DNA
＜213〉artificial sequence
<400>351
Claims (1)
1. the composite gene chip that detects of a food-borne pathogens, it is characterized in that: comprise eight parallel inferior arrays that make up with the detection probes group that detects each bacterium in eight kinds of food-borne pathogens, blank with delegation respectively between these eight inferior arrays as at interval, be configured to a big array;
Described eight kinds of food-borne pathogens are: Listeria monocytogenes, vibrio cholerae, Vibrio parahaemolyticus, Salmonellas, Shigellae, streptococcus aureus, campylobacter jejuni and escherichia coli;
Wherein, on the inferior array of Listeria monocytogenes 47 probes are arranged, sequence derives from 7 target genes shown in SEQ ID No.1-47; Have 93 on the inferior array of vibrio cholerae, sequence derives from 18 target genes shown in SEQ ID No.48-140; Have 33 on the inferior array of Vibrio parahaemolyticus, sequence derives from 9 target genes shown in SEQ IDNo.141-173; Have 31 on the inferior array of Salmonellas, sequence derives from 11 target genes shown in SEQID No.174-204; Have 31 on the inferior array of Shigellae, sequence derives from 8 target genes shown in SEQ ID No.205-235; Have 22 on the inferior array of streptococcus aureus, sequence derives from 9 target genes shown in SEQ ID No.236-257; Have 17 on the inferior array of campylobacter jejuni, sequence derives from 7 target genes shown in SEQ ID No.258-274; Have 73 on the inferior array of escherichia coli, sequence derives from 17 target genes shown in SEQ ID No.275-347;
Also comprise: 1 positive control probe, sequence is shown in SEQ ID No.348; 3 negative control probes, sequence are SEQ ID No.349-351.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101990395A CN101397586B (en) | 2008-10-10 | 2008-10-10 | Composite gene chip for food-borne pathogenic bacteria detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101990395A CN101397586B (en) | 2008-10-10 | 2008-10-10 | Composite gene chip for food-borne pathogenic bacteria detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101397586A CN101397586A (en) | 2009-04-01 |
| CN101397586B true CN101397586B (en) | 2011-02-02 |
Family
ID=40516429
Family Applications (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088111A (en) * | 2011-10-31 | 2013-05-08 | 中华人民共和国北京出入境检验检疫局 | F0F1-ATPase rotary molecular motor sensor kit for detecting Vibrio cholera |
| CN103088112A (en) * | 2011-10-31 | 2013-05-08 | 中华人民共和国北京出入境检验检疫局 | F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes |
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| CN101967510B (en) * | 2009-07-28 | 2012-10-17 | 天津生物芯片技术有限责任公司 | Gene chip and kit for detecting common pathogenic bacteria in food |
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| CN1384210A (en) * | 2002-05-14 | 2002-12-11 | 中国科学院武汉病毒研究所 | Detection method of enzyme labelling signal in gene chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103088111A (en) * | 2011-10-31 | 2013-05-08 | 中华人民共和国北京出入境检验检疫局 | F0F1-ATPase rotary molecular motor sensor kit for detecting Vibrio cholera |
| CN103088112A (en) * | 2011-10-31 | 2013-05-08 | 中华人民共和国北京出入境检验检疫局 | F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes |
| CN103088111B (en) * | 2011-10-31 | 2015-06-03 | 中华人民共和国北京出入境检验检疫局 | F0F1-ATPase rotary molecular motor sensor kit for detecting Vibrio cholera |
| CN103088112B (en) * | 2011-10-31 | 2015-06-03 | 中华人民共和国北京出入境检验检疫局 | F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes |
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