CN106591478A - Fluorescent PCR primers for detection of Saiga tatarica-derived ingredient and detection method using the same - Google Patents

Abstract

The invention discloses fluorescent PCR primers for detection of a Saiga tatarica-derived ingredient and a detection method using the same. According to a mitochondrial genomic DNA conserved sequence of Saiga tatarica, a pair of specific primers and probes are designed, and a Taqman probe fluorescent PCR method for detecting a Saiga tatarica-derived ingredient is built. A detection result shows that the designed primers/probes and detection method have good specificity, high sensitivity and strong stability. The fluorescent PCR primers play a key role in a forensic identification process in suspected Saiga tatarica smuggling case handling based on a law-enforcing department.

Description

For detecting the fluorescence PCR primer and detection method of sahilite derived component

Technical field

The invention belongs to Molecular Detection field, and in particular to a kind of fluorescent PCR for detecting sahilite derived component Primer and detection method.

Background technology

Sahilite also known as saiga tatarica, big nose antelope, scientific name Saiga tatarica, are Mammalia Artiodactyla Dong Jiao sections The unique species of sahilite subordinate.Its antelope's horn because with it is clearing heat and detoxicating the effect of be widely used.Terminate in glacial period Before, sahilite was once distributed widely in England and was also distributed to the length and breadth of land area between Siberia, or even Alaska.It How careless Plain of the integrated distribution in the Central Asia afterwards.Since 20th century, because Traditional Chinese Medicine thinks have from the antelope's horn of sahilite Medical value, goat's horn is sold China and does medicinal material, and the quantity of sahilite has fallen sharply 95%, sahilite within Chinese territory All extinctions.Sahilite is put into《World Conservation Union》(IUCN) endangered species Red List in 2012 Ver3.1 --- pole is endangered (CR), forbids to hunt.In order to strengthen the protection to sahilite, its illegal trading is limited, set up a kind of The DNA detection methods of more particularly suitable sahilite derived component are required.

At present both at home and abroad for the report of the identification of animal sahilite derived component mainly has microscopy, routine The methods such as PCR, GC/MSD analysis.Liu Caiyong light microscopes, observe the micro-structural feature of antelope's horn medicinal material difference sample； Chen employs DNA bar code technology to diagnose sahilite angle and substitute；Xu Caiyong gas chromatography-mass spectrums-computer connection 9 kinds of fatty acid compositions are identified from antelope's horn with technology.Above-mentioned detection method meets to a certain extent inspection inspection The demand of epidemic disease.However, above method there is also respective deficiency.For example, Microscope examination cannot meet quick and high-volume The needs of detection, while the requirement to reviewer is higher, is difficult to standardize and standardizes；Standard PCR technology take it is long, and Other are also needed to operate；The qualitative scarce capacity of GC/MSD methods, speed is slow.Therefore it is highly desirable to set up a kind of quick, sensitivity The method of detection sahilite derived component.

The content of the invention

It is an object of the invention to provide a kind of fluorescence PCR primer and detection side for detecting sahilite derived component Method.

The technical solution used in the present invention is：

For detecting the fluorescence PCR primer and probe of sahilite derived component, it is the mitochondria according to sahilite Designed by DNA sequence dna.

The nucleotide sequence of the primer and probe is as follows：

F-Primer：5 '-CAATCCTCCGATCAATTCCT-3 ' (SEQ ID NO.1),

R-Primer：5 '-ACTAGAACTCAGAATAGGCAT-3 ' (SEQ ID NO.2),

Probe：FAM-AATCCTAGTCCTCATACCCTTGC-BHQ1(SEQ ID NO.3).

A kind of kit for detecting sahilite derived component, it includes above-mentioned fluorescence PCR primer and probe.

The detection method of sahilite derived component, comprises the steps：

(1) sample DNA is extracted；

(2) with sample DNA as template, using the fluorescence PCR primer in following table, probe fluorescent PCR amplification is carried out；

(3) according to amplification curve whether containing sahilite derived component in judgement sample.

Fluorescent quantitation reaction system parameter is as shown in the table：

Fluorescence PCR conditional parameter is as follows：

The invention has the beneficial effects as follows：The present invention is devised according to the mitochondrial genomes DNA conserved sequence of sahilite A pair of specific primers and probe, set up the Taqman fluorescence probe PCR methods of detection sahilite derived component.Testing result Show, the primer/probe and detection method designed by the present invention has good specificity, and sensitivity height, stability are strong.It is right Law enforcement agency can play critical effect in the forensic identification program for processing doubtful sahilite smuggling case.

Description of the drawings

Fig. 1 is sahilite sample P CR testing result (M：DNA Marker DL2000；1. sahilite；N. it is negative right According to)；

Fig. 2 is sahilite fluorescent PCR testing result；

Fig. 3 is specificity experiments result；

Fig. 4 is sensitivity experiment result；

Fig. 5 is first time stability experiment result；

Fig. 6 is second stability experiment result.

Specific embodiment

With reference to embodiment, the present invention is further illustrated, but is not limited thereto.

Molecular biology experiment technology employed in following examples include PCR amplifications, plasmid extraction, plasmid convert, DNA fragmentation connection, digestion, gel electrophoresis etc., if no special instructions, generally conventionally operate, and specifically can be found in《Molecule Cloning experimentation guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated, 2002, Beijing：Science Press), or according to the condition proposed by manufacturer.

The foundation of the fluorescence PCR detecting method of the sahilite derived component of embodiment 1

1st, sample collection and keeping

2 parts of sahilite angle sample, animal sample 14 parts of (tiger bone, pilose antler, standard mink skin, dog meats, bear gall, golden monkey, pigs Meat, rhinoceros horn, Yak Meat, common beef, buffalo meat, Carnis Bovis seu Bubali, chevon, meat of a sheep) it is Zhuhai inspection and quarantining for import/export Sample is retained in inspection and quarantine technique center animal quarantine laboratory by office.

2nd, real-time fluorescence PCR primer and probe are designed

Have the one of specificity according to the design of the highly conserved region of sahilite animal mitochondria DNA (mtDNA) sequence Set primer and fluorescence probe, are synthesized by Shanghai Hui Rui bio tech ltd.Primer sequence refers to table 1.

The sahilite derived component fluorescence PCR primer of table 1, probe sequence

3rd, the extraction of sample DNA

The extracting method of DNA is carried out according to the chapters and sections extracted with regard to tissue DNA in DNA extraction kit specification, is obtained The sample total DNA solution of purifying.

4th, sample Species estimation

With the sahilite sample DNA that extracts as template, using the specific primer of this experimental design, (primer sequence is detailed 1) be shown in Table carries out standard PCR amplification in PCR instrument to target sequence.PCR reaction systems are 25 μ L, refer to table 2, and reaction condition is detailed 3 are shown in Table, purpose fragment expands 35 circulations.Product all carries out 2% agarose gel electrophoresis, reclaims purpose fragment, send Beijing Liuhe Huada Genomics Technology Co., Ltd's Wuhan Company carries out TA cloning and sequencings, and sequencing result is entered with target gene Row is compared, and determines its Species origin.

The reaction system parameter of table 2

The reaction condition parameter of table 3

Experimental result is as shown in Figure 1：Sahilite tissue sample is successfully entered performing PCR amplification, and primer size is about 151bp, is consistent with expection.PCR primer Jing cloning and sequencing, confirms that sample is from sahilite in Fig. 1.

5th, Taqman fluorescence probes PCR reaction systems and condition

Fluorescent PCR amplification is carried out by template of sahilite sample DNA.The μ L of reaction system 25, refer to table 4 below.This test The quantitative fluorescent PCR initial reaction parameter of foundation includes that denaturation, denaturation, annealing extend three steps.Real-time fluorescence quantitative PCR is preliminary Response parameter is shown in Table 5, and experimental result is as shown in Figure 2.

The sahilite derived component fluorescent quantitation reaction system parameter of table 4

The sahilite derived component fluorescent quantitation reaction condition parameter of table 5

Fig. 2 shows, has confirmed that amplification curve occurs in the DNA for sahilite, Cycle threshold (Ct) value ＜ 18, Negative control has no amplification curve.

The specificity experiments of embodiment 2

Using the Taqman fluorescence probe PCR reaction systems and condition of embodiment 1 to 14 parts of samples in addition to sahilite (tiger bone, pilose antler, standard mink skin, dog meats, bear gall, golden monkey, pork, rhinoceros horn, Yak Meat, common beef, buffalo meat, Carnis Bovis seu Bubali, Chevon, meat of a sheep) DNA profiling expanded, to verify the special of designed primer pair sahilite derived component detection Property.Experimental result is as shown in Figure 3.

Understand that in addition to sahilite source property sample has obvious amplification curve, other samples are without amplification according to Fig. 3.

The sensitivity experiment of embodiment 3

Recombinant plasmid obtained by above-mentioned Jing cloning and sequencings is determined into plasmid concentration using ultraviolet specrophotometer, according to Avobenzene Jia Deluo constants are scaled the copy number of genes of interest, and with 10 times of gradient dilutions to 10copies/ μ L, the sun to gradient dilution Property control carry out Fluorescence PCR detection.The sensitivity of checking institute method for building up, experimental result is as shown in Figure 4.

Understand according to Fig. 4,9 concentration gradients (10⁹-10¹) positive plasmid in, 9 concentration (10⁹-10¹) all occur expanding Increase curve, therefore the least concentration that can detect that for 10copies/ μ L, the as sensitivity of the detection method.

The stability experiment of embodiment 4

With above-mentioned plasmid concentration as 10⁶Positive criteria product be template, use set up method to be tested twice, try Time interval is tested for 30 days, 20 repetitions are done in test every time.Calculate the difference between testing result, the method for building up to verify Stability, experimental result is as shown in Figure 5 and Figure 6.

From Fig. 5, Fig. 6, using 2010 pairs of Ct values tested twice of Microsoft Excel single factor test variance is carried out Analysis, as a result shows that the coefficient of variation for repeating test in 2 times batches is respectively 0.88% and 1.13%, and interassay coefficient of variation is 1.38%.

This research devises a pair of specific primers and spy according to the mitochondrial genomes DNA conserved sequence of sahilite Pin, sets up the Taqman fluorescence probe PCR methods of detection sahilite derived component.Testing result shows that the present invention is designed Primer/probe and detection method there is good specificity, and sensitivity is high, stability is strong.Doubtful is being processed to law enforcement agency Like critical effect can be played in the forensic identification program of sahilite smuggling case.

SEQUENCE LISTING

<110>Inspection ＆ Quarantine Technology Center of Zhuhai Entry-Exit Inspection ＆ Quarantine Bureau

<120>For detecting the fluorescence PCR primer and detection method of sahilite derived component

<130>

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213>Artificial sequence

<400> 1

caatcctccg atcaattcct 20

<210> 2

<211> 21

<212> DNA

<213>Artificial sequence

<400> 2

actagaactc agaataggca t 21

<210> 3

<211> 23

<212> DNA

<213>Artificial sequence

<400> 3

aatcctagtc ctcataccct tgc 23

Claims (5)

1. it is used to detect the fluorescence PCR primer and probe of sahilite derived component, it is characterised in that the primer and probe are According to designed by the mtdna sequence of sahilite.

2. fluorescence PCR primer according to claim 1 and probe, it is characterised in that the nucleotides of the primer and probe Sequence is as follows：

F-Primer：5 '-CAATCCTCCGATCAATTCCT-3 ',

R-Primer：5 '-ACTAGAACTCAGAATAGGCAT-3 ',

Probe：5’-AATCCTAGTCCTCATACCCTTGC-3’.

3. a kind of kit for detecting sahilite derived component, it includes that the fluorescent PCR described in claim 1 or 2 draws Thing and probe.

4. the detection method of sahilite derived component, comprises the steps：

(1) sample DNA is extracted；

(2) with sample DNA as template, using the fluorescence PCR primer described in claim 1 or 2, probe fluorescent PCR amplification is carried out；

(3) according to amplification curve whether containing sahilite derived component in judgement sample.

5. detection method according to claim 4, it is characterised in that the Fluorescence PCR conditional parameter is as follows：

。