CN104152572B

CN104152572B - Triple real-time fluorescence PCR method and kit for simultaneously detecting three streptococci

Info

Publication number: CN104152572B
Application number: CN201410425186.5A
Authority: CN
Inventors: 李丹丹; 徐义刚; 高慎阳; 蔡波
Original assignee: INSPECTION AND QUARANTINE TECHNOLOGY CENTER OF HAINAN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Current assignee: INSPECTION AND QUARANTINE TECHNOLOGY CENTER OF HAINAN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Priority date: 2014-08-27
Filing date: 2014-08-27
Publication date: 2020-05-29
Anticipated expiration: 2034-08-27
Also published as: CN104152572A

Abstract

The invention provides a triple real-time fluorescence PCR method and a kit for simultaneously detecting three streptococci, wherein the kit comprises: the detection primer pair of Streptococcus agalactiae is shown in sequence tables Seq ID No.1 and 2 and the probe is shown in sequence table Seq ID No.3, the detection primer pair of Streptococcus dysgalactiae is shown in sequence tables Seq ID No.4 and 5 and the probe Seq ID No.6, the detection primer pair of Streptococcus iniae is shown in sequence tables Seq ID No.7 and 8 and probe Seq ID No.9, three positive control products are shown in sequence tables Seq ID No.16, 17 and 18, and the negative control product (ddH)₂O) and PCR buffer solution, dNTP and Taq DNA Polymerase required by fluorescent real-time quantitative PCR amplification. The method is simple and rapid, and has good specificity.

Description

Triple real-time fluorescence PCR method and kit for simultaneously detecting three streptococci

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a triple real-time fluorescence PCR method and a kit for simultaneously detecting three streptococci.

Background

The genus Streptococcus (Streptococcus) is a genus with a large population, which has been found to be at least 60 species and subspecies, the species of pathogenic streptococcal pathogenic bacteria are not many in aquatic animals, and the streptococcus which has been isolated from cultured fishes includes streptococcus iniae (s.iniae), streptococcus difficile (s. diffucilis), streptococcus agalactiae (s. agalactiae), streptococcus miehei (s. mileri), streptococcus paramyxis (s.parauberis), and streptococcus dysgalactiae (s. dysgalaciae), however, the reported pathogenic streptococci are mainly Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae, can cause the attack of various sea and fresh water fishes, which occur in all the main fish culture countries in the world, the harm to the cultured fishes in temperate zone, tropical zone and subtropical zone is serious, and fish streptococcicosis is reported in 23 countries in 6 continents. At present, fish streptococcicosis becomes a disease distributed worldwide, and causes huge economic loss to the world aquaculture industry every year.

At present, the detection of streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae is realized by combining the conventional enrichment culture, biochemical identification and an automatic enzyme-linked fluorescence immunoassay method, and has the defects of complicated working procedures, long detection period, low detection sensitivity, low detection efficiency, universal false negative and the like. Therefore, the development of a detection method which is simple, convenient, rapid and accurate to operate, high in sensitivity and easy to popularize has important significance for early diagnosis and early prevention of fish streptococcicosis, improvement of aquatic product quality and guarantee of human health.

The Real-Time quantitative PCR technology (Real-Time quantitative Polymerase Chain Reaction, Real-Time PCR) is distinguished from many detection technologies, not only has the advantages of rapidness, simplicity, sensitivity and the like, but also has stronger specificity and higher automation degree due to double insurance of primers and probes compared with the conventional PCR method, realizes Real-Time online detection, does not need post-treatment on PCR amplification products in the PCR amplification process, effectively solves the problems of false positive and inaccurate quantification caused by PCR product pollution, and becomes an increasingly important detection means in the detection field. However, the fluorescent quantitative PCR technique can only detect one target.

The multiplex fluorescent quantitative PCR technology is a new technology developed on the basis of fluorescent quantitative PCR, and emphasizes that a plurality of targets are amplified simultaneously in the same reaction system. There have been several recent studies that apply multiplex quantitative PCR techniques to the field of bacterial detection. Andrea G, et al established Escherichia coli O157: H7, Salmonella and Listeria monocytogenes multiplex fluorescence PCR detection methods, Moniserrat E, et al established 5 kinds of Vibrio parahaemolyticus, Vibrio cholerae, etc. multiplex PCR detection methods in fish and aquatic products, and the results show that the multiplex PCR system is basically the same as the single amplification result in the aspects of sensitivity and specificity. However, no kit has been reported for simultaneous detection of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae.

Disclosure of Invention

Based on the defects, the invention aims to provide a multiple quantitative Taqman PCR method capable of simultaneously detecting three main pathogenic streptococcus including streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae of aquatic animals, and simultaneously provides a corresponding kit based on the method.

The purpose of the invention is realized by the following technology:

1. a primer pair and a probe for simultaneously detecting three streptococci by a real-time fluorescence PCR method,

streptococcus agalactiae:

the upper primer cpsF-FP is shown in a sequence table Seq ID No.1,

the lower primer cpsF-RP is shown in a sequence table Seq ID No.2,

the PROBE cpsF-PROBE is shown in a sequence table Seq ID No. 3;

streptococcus dysgalactiae:

the upper primer MIG-FP is shown as a sequence ID No.4 in a sequence table,

the lower primer MIG-RP is shown in a sequence table Seq ID No.5,

the PROBE MIG-PROBE is shown in a sequence table Seq ID No. 6;

streptococcus iniae:

the upper primer 16SrRNA-FP is shown in a sequence table Seq ID No.7,

the lower primer 16SrRNA-RP is shown in a sequence table Seq ID No.8,

the PROBE 16SrRNA-PROBE is shown in a sequence table Seq ID No. 9;

the 5' -labeled fluorescein of the cpsF-PROBE PROBE, MIG-PROBE PROBE and 16SrRNA-PROBE PROBE are different and are FAM, HEX and ROX respectively.

2. A kit containing the primer pair and the probe for simultaneously detecting the three streptococci by using the real-time fluorescence PCR method.

3. A PCR amplification primer pair for simultaneously detecting positive reference substances of three streptococci by using a real-time fluorescence PCR method,

streptococcus agalactiae:

the upper primer SA-F is shown in a sequence table Seq ID No.10,

the lower primer SA-R is shown as a sequence table Seq ID No. 11;

streptococcus dysgalactiae:

the upper primer SD-F is shown in a sequence table Seq ID No.12,

the lower primer SD-R is shown in a sequence table Seq ID No. 13;

streptococcus iniae:

the upper primer SI-F is shown in a sequence table Seq ID No.14,

the lower primer SI-R is shown in a sequence table Seq ID No. 15.

4. A positive reference substance for simultaneously detecting three streptococci by using a real-time fluorescence PCR method,

pMD-T-cpsF, as shown in sequence table Seq ID No. 16;

pMD-T-MIG, as shown in sequence table Seq ID No. 17;

pMD-T-16SrRNA is shown in a sequence table Seq ID No. 18.

5. The primer pair and the probe for simultaneously detecting the three streptococci by using the real-time fluorescent PCR method are as follows:

2×Quantitech Multiplex PCR NoRox Master Mix	12. 5μL
		5μM cpsF- FP	0.5μL
5μM MIG- RP	0.5μL
		5μM cpsF-PROBE	1μL
5μM MIG- FP	0.5μL
		5μM MIG- RP	0.5μL
5μM MIG-PROBE	1μL
		5μM 16SrRNA- FP	0.5μL
5μM 16SrRNA-RP	0.5μL
		5μM 16SrRNA-PROBE	1μL
ddH₂O	3.5μL
		pMD-T-cpsF	1μL
pMD-T-MIG	1μL
		pMD-T-16SrRNA	1μL

the amplification reaction conditions are as follows:

Step 1 95℃ 15min；

Step 2 94℃ 60s

Step 3 60℃ 60s

step 4 reading, Go to Step 2 for 40 cycles.

6. The preparation method of the positive control for simultaneously detecting the three streptococci by using the real-time fluorescent PCR method comprises the following steps:

(1) preparation of PCR template: extracting and purifying the genomic DNA of three streptococcus,

(2) selecting the streptococcus agalactiae cpsF gene as a target gene, wherein in the step (2), PCR reaction systems of the three target genes are as follows:

10×PCR Buffer	2.5μL
		dNTP (2.5mM for each)	2.0μL
upstream primer	1.0μL
		Downstream primer	1.0μL
TaqDNA Polymerase(5U/μL)	0.5μL
		DNA template	0.5μL
ddH₂O	17.5μL

The reaction conditions of PCR are as follows: pre-denaturation at 95 ℃ for 5 min; 15s at 94 ℃, 20s at 57.6 ℃ and 30s at 72 ℃ for 35 cycles; final extension at 72 ℃ for 10 min;

designing a PCR amplification primer of a positive control:

the upper primer SA-F is shown in a sequence table Seq ID No.10,

the lower primer SA-R is shown in a sequence table Seq ID No.11, and PCR amplification of a target gene is carried out;

selecting a streptococcus dysgalactiae MIG gene as a target gene, and designing a PCR amplification primer of a positive control: the upper primer SD-F is shown in a sequence table Seq ID No.12,

the lower primer SD-R is shown in a sequence table Seq ID No.13, and PCR amplification of a target gene is carried out;

selecting streptococcus iniae 16SrRNA gene as a target gene, and designing a PCR amplification primer of a positive control:

the upper primer SI-F is shown in a sequence table Seq ID No.14,

the lower primer SI-R is shown as a sequence ID No.15 in a sequence table, and PCR amplification of a target gene is carried out;

(3) preparing positive control substances pMD-T-cpsF, pMD-T-MIG and pMD-T-16 SrRNA;

in the step (3), the preparation process of the positive control comprises the following steps: and (3) connecting the PCR product obtained in the step (2) with a cloning vector pMD-19-T vector, transforming Escherichia coli competence JM109 to obtain a positive cloning strain, and preparing plasmids pMD-T-cpsF, pMD-T-MIG and pMD-T-16SrRNA as positive reference substances.

7. A kit for a triple real-time fluorescent PCR method for simultaneously detecting three streptococci comprises three pairs of primer pairs and three probes:

streptococcus agalactiae

The upper primer cpsF-FP is shown in a sequence table Seq ID No.1,

the lower primer cpsF-RP is shown in a sequence table Seq ID No.2,

the PROBE cpsF-PROBE is shown in a sequence table Seq ID No. 3;

streptococcus dysgalactiae

The upper primer MIG-FP is shown as a sequence ID No.4 in a sequence table,

the lower primer MIG-RP is shown in a sequence table Seq ID No.5,

the PROBE MIG-PROBE is shown in a sequence table Seq ID No. 6;

streptococcus iniae

The upper primer 16SrRNA-FP is shown in a sequence table Seq ID No.7,

the lower primer 16SrRNA-RP is shown in a sequence table Seq ID No.8,

the PROBE 16SrRNA-PROBE is shown in a sequence table Seq ID No. 9;

three positive controls: pMD-T-cpsF, as shown in Seq ID No.16 of the sequence Listing, pMD-T-MIG, as shown in Seq ID No.17 of the sequence Listing, pMD-T-16SrRNA, as shown in Seq ID No.18 of the sequence Listing, and a negative control (ddH)₂O) and PCR buffer solution, dNTP and Taq DNA Polymerase required by fluorescent real-time quantitative PCR amplification.

The invention respectively uses campylobacter jejuni (ATCC 33560), Escherichia coli O157: H7 (ATCC 35150), Listeria monocytogenes (ATCC 19111), Salmonella typhi (CMCC 50115), Shigella (ATCC 12022), Staphylococcus aureus (ATCC 29213), Yersinia enterocolitica (ATCC 9610), Vibrio cholerae (ATCC 51394), Vibrio vulnificus (ATCC 27562), Vibrio alginolyticus (ATCC 33787), Vibrio parahaemolyticus (ATCC 27519), Streptococcus agalactiae (ATCC 13813), Streptococcus dysgalactiae (ATCC 35666) and Streptococcus iniae (ATCC 29178) genome DNA to carry out experiments so as to verify the specificity of detection by the method. Experimental results show that the method can specifically detect streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae, and primers do not have cross reaction with other bacteria, so that the method has high detection specificity. The kit disclosed by the invention also has the advantages of rapidness, simplicity, accuracy, good repeatability, stability, reliability and practicability.

Drawings

FIG. 1 shows the establishment of triple real-time fluorescence PCR detection method for Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae.

FIG. 2 shows the results of the triple real-time fluorescence PCR assay for Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae for the sensitivity of Streptococcus agalactiae.

FIG. 3 shows the results of the triple real-time fluorescence PCR assay for Streptococcus dysgalactiae, Streptococcus dysgalactiae and Streptococcus iniae.

FIG. 4 shows the results of a triple real-time fluorescence PCR assay for Streptococcus iniae, Streptococcus iniae and Streptococcus iniae for the sensitivity of Streptococcus iniae, Streptococcus iniae and Streptococcus iniae.

FIG. 5 shows the results of a triple real-time fluorescence PCR assay for Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae for detecting the specificity of Streptococcus agalactiae.

FIG. 6 shows the results of a triple real-time fluorescence PCR detection method for Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae for detecting the specificity of Streptococcus dysgalactiae.

FIG. 7 shows the results of a triple real-time fluorescence PCR detection method for Streptococcus iniae, Streptococcus dysgalactiae and Streptococcus iniae.

Detailed Description

The present invention is described in further detail below by way of specific examples.

Example 1

Preparation of three Streptococcus DNA templates

Three streptococci [ Streptococcus agalactiae (ATCC 13813), Streptococcus dysgalactiae (ATCC 35666) and Streptococcus iniae (ATCC 29178) which are derived from American Type Culture Collection (ATCC) ] genomic DNAs were extracted and purified with reference to the TIAnma Bacteria DNA Kit specification, and the corresponding reagents were added in this order:

(1) taking 1.5mL of bacterial liquid, centrifuging for 1min at 10000r/min, removing supernatant, adding 200 mu L of buffer solution GA, and completely suspending thalli;

(2) adding 20 μ L protease K (20mg/mL), adding 220 μ L buffer solution GB, mixing, and performing water bath at 70 deg.C for 10 min;

(3) adding 220 μ L anhydrous ethanol, mixing thoroughly for 15s, centrifuging briefly, transferring the obtained solution (including flocculent precipitate) to adsorption column CB3, centrifuging at 12000r/min for 30s, and discarding the liquid in the collecting tube;

(4) adding 500 μ L buffer GD (containing anhydrous ethanol) into adsorption column CB3, centrifuging at 12000r/min for 30s, and discarding the liquid in the collection tube;

(5) adding 600 μ L of rinsing liquid PW (containing anhydrous ethanol) into adsorption column CB3, centrifuging at 12000r/min for 30s, and discarding the liquid in the collection tube;

(6) repeating the previous operation;

(7) putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000r/min for 2min, standing at room temperature for 2-5min, and airing the residual rinsing liquid;

(8) transferring the adsorption column CB3 into a new collection tube, adding 100 μ L buffer TE into the center of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12000r/min for 2min, and collecting DNA eluate.

Example 2

Preparation of Positive control

1. Design and Synthesis of PCR primers

The streptococcus agalactiae cpsF gene was selected as the target gene, the streptococcus dysgalactiae MIG gene as the target gene and the streptococcus iniae 16SrRNA gene as the target gene, and synthetic primer pairs were designed by BLAST analysis, as shown in table 1:

TABLE 1 PCR primer pairs for three streptococci

2. The primers SA-F/SA-R, SD-F/SD-R and SI-F/SI-R of the target genes of the three streptococci are used for respectively amplifying the cpsF gene, the MIG gene of the streptococcus agalactiae and the 16S rRNA gene of the streptococcus iniae by a PCR method. The reaction systems of three sections of gene PCR are as follows:

The reaction conditions of PCR were: pre-denaturation at 95 ℃ for 5 min; 15s at 94 ℃, 20s at 57.6 ℃ and 30s at 72 ℃ for 35 cycles; final extension at 72 ℃ for 10 min.

The PCR products were ligated with pMD-19-T vector, respectively, and competent E.coli JM109 was transformed to obtain recombinant bacteria, and recombinant plasmids were prepared according to the instructions of the Small plasmid DNA extraction kit of Shunhuan:

(1) selecting a single colony of the positive clone, inoculating the single colony in 5mL LB culture medium containing 100 mu g/mL Ampr, and culturing overnight at 37 ℃;

(2) taking 1-3mL of overnight culture liquid, centrifuging at 12000r/min for 5min, removing supernatant, adding 250 mu L of buffer P1 (containing RNase), and fully shaking thallus precipitates until the thallus precipitates are completely suspended;

(3) adding 250 μ L of Buffer P2, immediately and gently inverting the centrifuge tube from top to bottom for 6-10 times, mixing, and standing at room temperature for 2-4 min;

(4) adding 350 μ L Buffer P3, gently and repeatedly inverting the centrifuge tube for 6-10 times, mixing, and centrifuging at 12000r/min for 10 min;

(5) transferring the supernatant into an adsorption column, centrifuging at 12000r/min for 30s, discarding the filtrate, and placing the adsorption column into a collection tube;

(6) adding 500 mu L B1 liquid, centrifuging at 12000r/min for 30s, discarding the filtrate, and placing the adsorption column into the collection tube;

(7) adding 500 μ L W1 solution (containing anhydrous ethanol), centrifuging at 12000r/min for 30s, discarding filtrate, and placing adsorption column into collection tube;

(8) adding 500 μ L W1 solution (containing anhydrous ethanol), standing at room temperature for 1min, centrifuging at 12000r/min for 30s, discarding filtrate, placing adsorption column into collection tube, and centrifuging at 12000r/min for 1 min;

(9) transferring the adsorption column into a clean 1.5mL centrifuge tube, adding 150 μ L deionized water into the center of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000r/min for 1min, eluting, and collecting plasmid DNA.

The correctly identified recombinant plasmids were named pMD-T-cpsF, pMD-T-MIG, pMD-T-16SrRNA, respectively.

And quantifying the extracted plasmid DNA, and storing the quantified plasmid DNA at-20 ℃ for later use as a positive quality control standard substance of the kit. The quantitative calculation formula is: positive plasmid copy number copies/. mu.L = (OD)₂₆₀×50×10^-9X dilution multiple x 6.02 x 10²³) (660X number of bases), wherein: 50 represents the measurement in OD using a cuvette with a diameter of 1cm₂₆₀The corresponding double-stranded DNA concentration at 1 is 50. mu.g/mL; 660 represents the double-stranded DNA base pair average molecular weight.

Example 3

Design synthesis of triple real-time fluorescent PCR (polymerase chain reaction) primers and probes for three streptococcus

According to the genomic DNA sequences of streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae published in Genbank, by combining bioinformatics means, sequences of conserved regions of cpsF gene of streptococcus agalactiae, MIG gene of streptococcus dysgalactiae and 16S rRNA gene of streptococcus iniae are determined as detection target sequences, and 3 pairs of primers and 3 probes are designed and synthesized so as to carry out qualitative detection on streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae. As shown in table 2.

TABLE 2 triple real-time fluorescent PCR primers and probes for three streptococci

Example 4

Optimization of triple fluorescent PCR conditions

The invention adopts a single-factor concentration gradient experiment method to change the concentration of each component in the real-time fluorescent PCR reaction system one by one so as to optimize the real-time fluorescent PCR reaction system and establish triple real-time fluorescent PCR. The optimization results are shown in fig. 1. The optimized PCR reaction system is as follows:

the amplification reaction conditions are as follows:

Step 1 95℃ 15min；

Step 2 94℃ 60s

Step 3 60℃ 60s

step 4 reading, Go to Step 2 for 40 cycles.

Example 5

Sensitivity test for detecting three kinds of streptococcus by triple fluorescence PCR

Respectively to a concentration of about 1.52X 10⁸Streptococcus agalactiae, at a concentration of about 1.33X 10⁸Streptococcus dysgalactiae, at a concentration of about 1.76X 10⁷CFU/mL Streptococcus iniae were diluted in 10-fold gradients, 3 per concentration gradient in parallel. And extracting the genome DNA of each dilution of streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae by using the kit, and performing real-time fluorescence PCR detection on the streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae by using the extracted genome DNA as a template to determine the sensitivity of the detection method. The results are shown in FIGS. 2-4. As can be seen, the original bacterial liquid of Streptococcus agalactiae was diluted to 10^-7Can still be detected, and shows that the detection sensitivity of the detection method to the streptococcus agalactiae is about 1.52 multiplied by 10²CFU/mL (see FIG. 2); diluting original bacteria liquid of streptococcus dysgalactiae to 10^-6Can still be detected, which shows that the detection sensitivity of the detection method to the streptococcus dysgalactiae is about 1.33 multiplied by 10²CFU/mL (see FIG. 3); diluting original bacterial liquid of streptococcus iniae to 10^-6Can still be detected, which shows that the detection sensitivity of the detection method to the streptococcus iniae is about 1.76 multiplied by 10²CFU/mL (see FIG. 4).

Example 6

Triple fluorescence PCR detection test for specificity of three streptococci

The bacteria in table 3 were detected by the established triple real-time fluorescent PCR detection method to explore the specificity of detection of the method, and the results are shown in table 1. The results show that the method of the invention is capable of specifically detecting Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae without cross-reacting with other strains, indicating that the method of the invention has high specificity, as shown in FIGS. 5-7.

TABLE 3 specific results of real-time fluorescent PCR method

Bacterial strains	Origin of origin	Fluorescent PCR results
			Campylobacter jejuniCampylobacter jejuni	ATCC 33560	－
Enterohemorrhagic Escherichia coliEnterohemorrhagic E.coli O₁₅₇: H	ATCC 35150	－
			Listeria monocytogenesListeria monocytogenes	ATCC 19111	－
Salmonella typhosaSalmonella typhimurium	CMCC50115	－
			ShigellaShigella	ATCC 12022	－
Staphylococcus aureusStaphylococcus aureus	ATCC 29213	－
			Yersinia enterocoliticaYersinia enteroxolitica	ATCC 9610	－
Vibrio choleraeVibrio cholerae	ATCC 51394	－
			Vibrio vulnificusVibrio vulnificus	ATCC 27562	－
Vibrio alginolyticusVibrio alginolyticus	ATCC 33787	－
			Vibrio parahaemolyticusVibrio parahaemolyticus	ATCC 27519	－
Streptococcus agalactiaeStreptococcus agalactiae	ATCC 35666	+
			Streptococcus dysgalactiaeStreptococcus dysgalactiae	ATCC 13813	+
Streptococcus iniaeStreptococcus iniae	ATCC29178	+

Example 7

Triple fluorescence PCR (polymerase chain reaction) repeatability test for detecting three streptococci

1. The method is utilized to repeatedly detect the same positive standard substance for 20 times, and the repeatability and the stability of the detection of the method are inspected. The results show that the detection results are the same every time.

2. The method provided by the invention is used for detecting the positive standard substance of the same batch at intervals of 30 days, and the repeatability and stability of the detection of the method are inspected. The results show that the results of the two tests are the same.

Example 8

Triple fluorescence PCR detection three streptococcus practice verification test

The established triple real-time fluorescence PCR detection method is applied to detect the collected actual samples, 181 parts of tilapia sample are detected together, 2 parts of streptococcus iniae positive samples are detected as a result, the single real-time fluorescence PCR detection method is used for verification, the coincidence rate is 100%, and the method has good reliability and practicability.

<110> inspection and quarantine technology center of Hainan entry-exit inspection and quarantine bureau of people's republic of China

<120> triple real-time fluorescence PCR method and kit for simultaneously detecting three streptococci

<160>18

<210>1

<211>22

<212>DNA

<213> Artificial sequence

<400>1

TGAAAATTTG TCTGGTTGGT TC

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<400>2

CGGCACCAGA TGATATGATA AC

<210>3

<211>27

<212>DNA

<213> Artificial sequence

<400>3

TGGTGGTCAT CTAGCACACT TGAACCT

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<400>4

AAATGATTTA GTTGCGACTG AC

<210>5

<211>24

<212>DNA

<213> Artificial sequence

<400>5

TTCAACAATC CTATCTTGAG AATC

<210>6

<211>26

<212>DNA

<213> Artificial sequence

<400>6

TGCTACTCCG GGAGGCGTAT TTACAG

<210>7

<211>22

<212>DNA

<213> Artificial sequence

<400>7

GCGTTGTATT AGCTAGTTGG TG

<210>8

<211>19

<212>DNA

<213> Artificial sequence

<400>8

AAACCTTCTT CACTCACGC

<210>9

<211>26

<212>DNA

<213> Artificial sequence

<400>9

ACGGCTCACC AAGGCGACGA TACATA

<210>10

<211>21

<212>DNA

<213> Artificial sequence

<400>10

ATGAAAATTT GTCTGGTTGG T

<210>11

<211>23

<212>DNA

<213> Artificial sequence

<400>11

ATTCCTCCTA AATTAATTGC CTT

<210>12

<211>21

<212>DNA

<213> Artificial sequence

<400>12

CTTTTGGATT AGCGTCTGTA T

<210>13

<211>21

<212>DNA

<213> Artificial sequence

<400>13

TCTTTACGTT TTGAAGCGAC T

<210>14

<211>21

<212>DNA

<213> Artificial sequence

<400>14

GTAGAACGCT GAGGATTGGT G

<210>15

<211>21

<212>DNA

<213> Artificial sequence

<400>15

ATCTAATCCT GTTTGCTCCC C

<210>16

<211>3135

<212>DNA

<213> Artificial sequence

<400>16

TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 60

CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 120

TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC 180

ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC 240

ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300

TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT 360

TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT ACCCGGGGAT 420

CCTCTAGAGA TATGAAAATT TGTCTGGTTG GTTCAAGTGG TGGTCATCTA GCACACTTGA 480

ACCTTTTGAA ACCCATTTGG GAAAAAGAAG ATAGGTTTTG GGTAACCTTT GATAAAGAAG 540

ATGCTAGGAG TATTCTAAGA GAAGAGATTG TATATCATTG CTTCTTTCCA ACAAACCGTA 600

ATGTCAAAAA CTTGGTAAAA AATACTATTC TAGCTTTTAA GGTCCTTAGA AAAGAAAGAC 660

CAGATGTTAT CATATCATCT GGTGCCGCTG TAGCAGTACC ATTCTTTTAT ATTGGTAAGT 720

TATTTGGTTG TAAGACCGTT TATATAGAGG TTTTCGACAG GATAGATAAA CCAACTTTGA 780

CAGGAAAATT AGTGTATCCT GTAACAGATA AATTTATTGT TCAGTGGGAA GAAATGAAAA 840

AAGTTTATCC TAAGGCAATT AATTTAGGAG GAATATCGTC GACCTGCAGG CATGCAAGCT 900

TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCAC 960

ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC 1020

TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC 1080

TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG 1140

CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC 1200

ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT 1260

GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC 1320

ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA 1380

ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC 1440

CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG 1500

CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC 1560

TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC 1620

GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA 1680

GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT 1740

ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG 1800

GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT 1860

TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT 1920

TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA 1980

GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA 2040

TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC 2100

CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA 2160

TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC 2220

CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA 2280

GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA 2340

GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG 2400

TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC 2460

GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG 2520

TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT 2580

CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT 2640

CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA 2700

ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC 2760

GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC 2820

CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA 2880

GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT 2940

TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT 3000

TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC 3060

CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA 3120

CGAGGCCCTT TCGTC 3135

<210>17

<211>4654

<212>DNA

<213> Artificial sequence

<400>17

TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 60

CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 120

TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC 180

ACCATATGCGGTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC 240

ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300

TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT 360

TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT ACCCGGGGAT 420

CCTCTAGAGA TCTTTTTACG TAAATCAGCT TTTGGATTAG CGTCTGTATC AGCTGCGTTT 480

TTAGTTTCGG GAGCACTAGA AAATACTATA ACTGTTTCTG CAGAAACTAT ACCTGCAGCG 540

GTCATTGTAC CTGTTGGCCT AGATACTACA GAATTACAAA AATGGTATGA CATTGCAAAT 600

GATTTAGTTG CGACTGACAA TGCTACTCCG GGAGGCGTAT TTACAGCAGA CTCAATGAAG 660

GCATTATATC GTTTACTAAA TGATGCATAC GATGTGTTGG AATCAAAAGA CTATAGAAAA 720

TATGATTCTC AAGATAGGAT TGTTGAATTG GTAAACAATT TAAAGAATAC TACGCAGTCT 780

CTTTTACCAA TTGGAGTAGA ACCAGTAGTA TTTGATACTA CTCGCTTGAA TACCTGGTAT 840

GATGCTGCTA ATGAAATTGT TAATAATTCA GATGCTTATA CAGCAGAATC AATTCAGCCG 900

TTGTATAAGT TAATTAATGA TGCATACGAT GTGTTAGAAT CAAAAGATTA CAGTAAGTAT 960

GATTCTCAAG ATAAAGTCAA CAATCTTGCA GATCAGTTGA GAGATGCAGT TCAGGCAGTT 1020

CAACTAGAAG CACCTACAGT GATTGACGCA CCTGAACTAA CTCCAGCTTT GACTACTTAC 1080

AAACTTGTTG TTAAAGGTAA CACTTTCTCA GGAGAAACAA CTACTAAAGC CATCGATACT 1140

GCAACTGCGG AAAAAGAATT CAAACAATAC GCAACAGCTA ACAATGTTGA CGGTGAGTGG 1200

TCTTATGACG ATGCAACTAA AACCTTTACA GTTACTGAAA AACCAGCAGT GATTGACGCA 1260

CCTGAACTAA CTCCAGCCTT GACTACTTAC AAACTTATTG TTAAAGGTAA CACTTTCTCA 1320

GGCGAAACAA CTACTAAAGC AGTAGACGCA GAAACTGCAG AAAAAGCCTT CAAACAATAC 1380

GCAACAGCTA ACAATGTTGA CGGTGAGTGG TCTTATGACG ATGCAACTAA AACCTTTACA 1440

GTTACTGAAA AACCAGCAGT GATTGACGCA CCTGAACTAA CTCCAGCCTT GACTACTTAC 1500

AAACTTATTG TTAAAGGTAA CACTTTCTCA GGCGAAACAA CTACTAAAGC TATCGATGCT 1560

GCAACTGCAG AAAAAGAATT CAAACAATAC GCAACAGCTA ACGGTGTTGA CGGTGAATGG 1620

TCTTATGACG ATGCAACTAA AACCTTTACA GTTACTGAAA AACCAGCAGT GATTGACGCA 1680

CCTGAACTAA CTCCAGCCTT GACTACTTAC AAACTTATTG TTAAAGGTAA CACTTTCTCA 1740

GGCGAAACAA CTACTAAAGC AGTAGACGCA GAAACTGCAG AAAAAGCCTT CAAACAATAC 1800

GCTAACGAAA ACGGTGTTTA CGGTGAATGG TCTTATGACG ATGCAACTAA AACCTTTACA 1860

GTTACTGAAA AACCAGCAGT GATTGACGCA CCTGAATTAA CACCAGCATT GACAACCTAC 1920

AAACTTGTTA TCAATGGTAA AACATTGAAA GGCGAAACAA CTACTAAAGC AGTAGACGCA 1980

GAAACTGCAG AAAAAGCCTT CAAACAATAC GCTAACGAAA ACGGTGTTGA TGGTGTTTGG 2040

ACTTACGATG ATGCGACTAA GACCTTTACG GTAACTGAAA TGGTTACTGA AGTTCCTGGT 2100

GATGCACCAA CTGAACCAGA AAAGCCAGAA GCAAGTATCC CTCTTGTTCC GTTAACTCCT 2160

GCAACTCCAA TTGCTAAAGA TGACGCTAAG AAAGACGATA CTAAGAAAGT CGATACTAAG 2220

AAAGAAGACG CTAAAAAACC AGAAGCTAAG AAAGAAGAAG CTAAGAAAGA AGAAGCTAAG 2280

AAAGCTGCAACTCTTCCTAC AACTGGTGAA GGAAGCAACC CATTTTTCAC AGCTGCTGCG 2340

CTTGCAGTAA TGGCTGGTGC GGGTGCTTTG GCAGTCGCTT CAAAACGTAA AGAATCGTCG 2400

ACCTGCAGGC ATGCAAGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT GTGAAATTGT 2460

TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT 2520

GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC TTTCCAGTCG 2580

GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG 2640

CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT CGTTCGGCTG 2700

CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT 2760

AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC 2820

GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC 2880

TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA 2940

AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT 3000

CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG 3060

TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC 3120

GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG 3180

GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC 3240

TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT CTGCGCTCTG 3300

CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC 3360

GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT 3420

CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT 3480

TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA 3540

AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA 3600

TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC 3660

TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT 3720

GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA 3780

GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT 3840

AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT 3900

GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC 3960

GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC 4020

TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT 4080

ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT 4140

GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC 4200

CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT 4260

GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG 4320

ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT 4380

GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA 4440

TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA GGGTTATTGT 4500

CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC 4560

ACATTTCCCC GAAAAGTGCC ACCTGACGTC TAAGAAACCA TTATTATCAT GACATTAACC 4620

TATAAAAATA GGCGTATCAC GAGGCCCTTT CGTC 4654

<210>18

<211>3431

<212>DNA

<213> Artificial sequence

<400>18

TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 60

CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 120

TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC 180

ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC 240

ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300

TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT 360

TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT ACCCGGGGAT 420

CCTCTAGAGA TGTAGAACGC TGAGGATTGG TGCTTGCACT AATCCAAAGA GTTGCGAACG 480

GGTGAGTAAC GCGTAGGTAA CCTACCTCAT AGCGGGGGAT AACTATTGGA AACGATAGCT 540

AATACCGCAT GANACTAGAG TACACATGTA CTTAATTTAA AAGGAGCAAT TGCTTCACTA 600

TGAGATGGAC CTGCGTTGTA TTAGCTAGTT GGTGAGGTAA CGGCTCACCA AGGCGACGAT 660

ACATAGCCGA CCTGAGAGGG TGATCGGCCA CACTGGGACT GAGACACGGC CCANACTCCT 720

ACGGGAGGCA GCAGTAGGGA ATCTTCGGCA ATGGACGGAA GTCTGACCGA GCAACGCCGC 780

GTGAGTGAAG AAGGTTTTCN GATCGTAAAG CTCTGTTGTT AGAGAAGAAC GGTAATGGGA 840

GTGGAAAATC CATTACGTGA CGGTAACTAA CCAGAAAGGG ACGGCTAACT ACGTGCCAGC 900

AGCCGCGGTA ATACGTANGT CTCGAGCGTT GTCCGGATTT ATTGGGCGTA AAGCGAGCGC 960

AGGCGGTTCT ATAAGTCTGA AGTAAAAGGC AGTGGCTCAA CCATTGTATG CTTTGGAAAC 1020

TGTAGAACTT GAGTGCAGAA GGGGAGAGTG GAATTCCATG TGTAGCGGTG AAATGCGTAN 1080

ATATATGGAG GAACACCGGT GGCGAANGCG GCTCTCTGGT CTGTAACTGA CGCTGAGGCT 1140

CGAAAGCGTG GGGAGCAAAC AGGATTAGAT ATCGTCGACC TGCAGGCATG CAAGCTTGGC 1200

GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA 1260

CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC 1320

ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA AACCTGTCGT GCCAGCTGCA 1380

TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC 1440

CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT CAGCTCACTC 1500

AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC GCAGGAAAGA ACATGTGAGC 1560

AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG 1620

GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC 1680

GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT 1740

TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT 1800

TTCTCATAGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG 1860

CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT 1920

TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT 1980

TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC CTAACTACGG 2040

CTACACTAGA AGAACAGTAT TTGGTATCTG CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA 2100

AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT GGTAGCGGTG GTTTTTTTGT 2160

TTGCAAGCAG CAGATTACGC GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC 2220

TACGGGGTCT GACGCTCAGT GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT 2280

ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA TGAAGTTTTA AATCAATCTA 2340

AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC TTAATCAGTG AGGCACCTAT 2400

CTCAGCGATC TGTCTATTTC GTTCATCCAT AGTTGCCTGA CTCCCCGTCG TGTAGATAAC 2460

TACGATACGG GAGGGCTTAC CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG 2520

CTCACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG 2580

TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG AAGCTAGAGT 2640

AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTACAG GCATCGTGGT 2700

GTCACGCTCG TCGTTTGGTA TGGCTTCATT CAGCTCCGGT TCCCAACGAT CAAGGCGAGT 2760

TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC CGATCGTTGT 2820

CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG GCAGCACTGC ATAATTCTCT 2880

TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTACTCAA CCAAGTCATT 2940

CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG GCGTCAATAC GGGATAATAC 3000

CGCGCCACAT AGCAGAACTT TAAAAGTGCT CATCATTGGA AAACGTTCTT CGGGGCGAAA 3060

ACTCTCAAGG ATCTTACCGC TGTTGAGATC CAGTTCGATG TAACCCACTC GTGCACCCAA 3120

CTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA 3180

AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT 3240

TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC ATGAGCGGAT ACATATTTGA 3300

ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC 3360

TGACGTCTAA GAAACCATTA TTATCATGAC ATTAACCTAT AAAAATAGGC GTATCACGAG 3420

GCCCTTTCGT C 3431

Claims

1. A triple real-time fluorescence PCR detection kit for simultaneously detecting three streptococci, wherein the three streptococci are streptococcus agalactiae, streptococcus dysgalactiae and streptococcus iniae, and the kit is characterized by comprising: primers and probes for amplifying three streptococci, three positive control substances and negative control substance ddH₂O, PCR buffer solution, dNTP and Taq DNA polymerase;

the three positive control substances are respectively:

pMD-T-cpsF, as shown in sequence table Seq ID No.16,

pMD-T-MIG is shown in a sequence table Seq ID No.17,

pMD-T-16SrRNA is shown in a sequence table Seq ID No.18,

the streptococcus agalactiae amplification primers and the probes are respectively as follows:

the upper primer cpsF-FP is shown in a sequence table Seq ID No.1,

the lower primer cpsF-RP is shown in a sequence table Seq ID No.2,

the PROBE cpsF-PROBE is shown in a sequence table Seq ID No. 3;

the streptococcus dysgalactiae amplification primers and the probes are respectively as follows:

the upper primer MIG-FP is shown as a sequence ID No.4 in a sequence table,

the lower primer MIG-RP is shown in a sequence table Seq ID No.5,

the PROBE MIG-PROBE is shown in a sequence table Seq ID No. 6;

the streptococcus iniae amplification primer and the probe are respectively as follows:

the upper primer 16SrRNA-FP is shown in a sequence table Seq ID No.7,

the lower primer 16SrRNA-RP is shown in a sequence table Seq ID No.8,

the PROBE 16SrRNA-PROBE is shown in a sequence table Seq ID No. 9.