CN102242191B

CN102242191B - PCR (polymerase chain reaction) primer for detecting microcystins in water bodies

Info

Publication number: CN102242191B
Application number: CN2011101114415A
Authority: CN
Inventors: 朱鹏; 严小军; 严婷婷; 陈海敏; 游玉容
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2011-04-25
Filing date: 2011-04-25
Publication date: 2013-12-04
Anticipated expiration: 2031-04-25
Also published as: CN102242191A

Abstract

The invention discloses a PCR (polymerase chain reaction) primer for detecting microcystins in water bodies. According to different regions in a biosynthetic pathway of microcystins, 6 pairs of specific primers are designed. The invention also discloses application of the PCR primer. The specific evaluation analysis shows that the result of detecting microcystins by using the 6 pairs of primers is consistent with the result of detecting microcystins by using an LC-MS (liquid chromatograph-mass spectrometer), and the PCR amplification result shows that stripes are single. Compared with the traditional detection of microcystins in water bodies, by adopting the mature PCR technology, the 6 pairs of primers can quickly and sensitively detect whether microcystins exist in water bodies or not, thereby greatly improving the detection sensitivity and efficiency.

Description

PCR primer sets for detection of Microcystins in Water

Technical field

The present invention relates to a kind of primer of the PCR for detection of Microcystins in Water, the invention still further relates to this PCR primer and detect the application of Microcystin in water body.

Background technology

Blue-green algae has another name called " blue-green algae ", at some in nutritious water body, some blue-green algae often in summer amount reproduction form " wawter bloom ".When causing water quality deterioration, some " wawter bloom " kind also can produce the secondary metabolite with bio-toxicity, is called " cyanophycean toxin ".The release of these cyanophycean toxins directly produces harm to fish, people and animals.The difference of the illness that cyanophycean toxin finally causes according to it can be divided into four large classes: neurotoxin, hepatotoxin, cytotoxin and pungency toxin (irritant toxins).In this four toxoid, hepatotoxin is the Health hazard maximum of Microcystin (microcystin) to the public, and serious meeting causes its death.Therefore, around how detecting, controlling and eliminate cyanophycean toxin, the scientific worker of various countries has carried out a large amount of research work.

For the detection of Microcystin, carry out toxicity tests in the detection water body, whether to have cyanophycean toxin to exist by building corresponding living model in early days.Yet a series of ethics problems that lower sensitivity, higher experiment expend and toxicity tests brings, make people have to select method for distinguishing to be diagnosed cyanophycean toxin.Along with the micro-example analytical instrument (as, HPLC, MALDI-TOF) development, rely on these instrument developments to go out the multiple method for detection of cyanophycean toxin in natural water.These methods analyst process simple and fasts and there is very high sensitivity.Yet, because analytical instrument has very high requirement to the pre-treatment of sample, therefore in practice, the processing of the sample not only time-consuming but also effort that becomes, and expensive analytical instrument and standard substance also make many laboratories can't carry out the diagnosis to cyanophycean toxin.Simultaneously, the diagnostic means that relies on immunology and biochemical method to develop also applies to the detection of cyanophycean toxin in laboratory or physical environment water body gradually.As, the ELISA method that hepatotoxin (microcystin and nodularin) is detected, colorimetry (protein phosphatase inhibitor) and cytotoxicity diagnosis.

Yet, aforementioned multiple diagnostic method, the Microcystin be released in water body of all take is detected object, can not before being discharged into water body in frustule, to it, judge in advance at toxin.Can't carry out preliminary examination to potential product poison blue-green algae by these methods.With respect to traditional Microcystin detection technique, use ripe round pcr in conjunction with efficient, the special primer for the Microcystin synthetic gene can be quick, whether exist in sensitive detection water body and produce virus gene and then can determine in water body whether contain maybe and may produce Microcystin, thereby also improved the anticipation power that toxin detects when increasing the sensitivity detected.Total institute is known, and the key of PCR detection technique is the specificity of primer, and good Auele Specific Primer can, so that the PCR result is clear, band is single, can also be avoided false-positive generation simultaneously.Therefore, the design of primer applies to Microcystin for success by round pcr with invention and detects just show particularly important.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of primer of the PCR for detection of Microcystins in Water for the above-mentioned state of the art, for different zones in the Microcystin biosynthetic pathway, has designed altogether 6 pairs of Auele Specific Primers.

In the biological synthesis gene cluster of Microcystin, mcyA, mcyB, mcyC, mcyD, these 6 main large fragment gene pairs Microcystins of mcyE, mcyG synthetic has very important effect.On the other hand, the malicious Microcystis aeruginosa of non-product also may contain part product virus gene.Therefore, we have designed 6 pairs of primers for Microcystin synthetic gene bunch.Get rid of false-positive generation with the mutual rectification by multipair primer, also make 6 pairs of primers there is identical annealing temperature, can carry out pcr amplification simultaneously, improve amplification efficiency simultaneously.

The design concept of primer: for the A structural domain design synthetic primer of the Ala-D of the mcyA genes encoding in the Microcystin gene cluster and Mdha to MCYAAAF MCYAAAR and MCYAMAF MCYAMAR; For the A structural domain design synthetic primer of the D-MeAsp of mcyB genes encoding to MCYBAF MCYBAR; For the A structural domain design synthetic primer of the D-Glu of mcyE genes encoding to MCYEAF MCYEAR; The A structural domain of the Adda of mcyG genes encoding design synthetic primer to MCYGAF MCYGAR; For the KSdomain of mcyE genes encoding, the KS domain of mcyG genes encoding, the KS domain1 of mcyD genes encoding and KS domain2 synthetic primer to MCYKSF MCYKSR.

The details of 6 pairs of primers:

Compared with prior art, the invention has the advantages that: show by the specificity analysis and assessment, apply the Microcystin detected result that 6 pairs of primers carry out consistent with the LC-MS detected result, and pcr amplification band is single as a result.Simultaneously, 6 pairs of primers have identical annealing temperature, can carry out pcr amplification simultaneously, avoid due to single, primer being detected and forming false-positive phenomenon.In sum, with respect to the detection of Microcystin traditional in water body, use these 6 pairs of primers of ripe round pcr can be quick, in sensitive detection water body, whether have Microcystin, thereby greatly improve sensitivity and the efficiency detected.

The accompanying drawing explanation

Fig. 1 be in embodiment primer sets MCYAMAF the pcr amplification result of MCYAMAR.

Fig. 2 be in embodiment primer sets MCYBAF the pcr amplification result of MCYBAR

Fig. 3 be in embodiment primer sets MCYGAF the pcr amplification result of MCYGAR.

Fig. 4 be in embodiment primer sets MCYAAAF the pcr amplification result of MCYAAAR.

Fig. 5 be in embodiment primer sets MCYEAF the pcr amplification result of MCYEAR

Fig. 6 be in embodiment primer sets MCYKSF the pcr amplification result of MCYKSR.

Embodiment

Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.

Embodiment:

1, algal species cultivation: from Chinese Academy of Sciences typical case culture collection council's algae kind storehouse (FACHB Collection), buy ten strain algae kinds, 3 strain Microcystis aeruginosa (MA-1, MA-2, MA-3) wherein, 2 strain Microcystis-flos-aquae (MF-2, MF-3), 2 strain Microcystis wesenbergii (MW-1, MW-2), 1 strain Microcystis elabens (ME-1), 1 strain Microcystis viridis (MV-1), 1 strain Microcystisichy0blabe (MI-1).Adopt the BG11 liquid nutrient medium to cultivate, 25 ℃ of room temps, intensity of illumination is 1000-1500lux, and Light To Dark Ratio is 12: 12, and every day, shaking flask was 1-2 time, standing cultivation.

2, toxin analysis: adopt liquid chromatography-triple quadrupole bar mass spectrograph (LC-MS) to make toxin analysis, analytical results (table 1):

The LC-MS analytical results of table 10 strain Microcystins

Annotate: in table, MCYST is microcystin, and " no " means not detect toxin

3, the PCR of toxin synthetic gene bunch detects: with 6 couple of our design, for the primer pair ten strain Microcystis aeruginosas of Microcystin synthetic gene bunch design, carry out pcr amplification.The PCR reaction system is 25 μ L systems.Toxin synthetic gene PCR reaction conditions is: 94 ℃ of min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 30s; 30 circulations; 72 ℃ of 3min.The PCR product carries out 1% agarose gel electrophoresis, and as shown in Fig. 1～Fig. 6, the PCR band is special and single.M:Marker2000 molecular weight standard in Fig. 1, Fig. 2, Fig. 3, Fig. 5 and Fig. 6 wherein, 1-11 is respectively: MF-2, MF-3, MI-1, MA-1, MW-1, ME-1, MA-2, MA-3, MW-2, MV-1, blank.M:Marker2000 molecular weight standard in Fig. 4,1-11 is respectively: MF-2, MF-3, MI-1, MA-1, MW-1, MW-2, MA-2, MA-3, ME-1, MV-1, blank.

4, the comparison of PCR result and LC-MS analytical results: by PCR result and LC-MS analytical results are compared, we find the amplification of 6 pairs of primers and the consistence (table 2) that the LC-MS analytical results has height.

Table 2 toxin gene pcr amplification result and LC-MS result are relatively

Annotate: "-": mean that result is negative, "+": be expressed as the positive.

From above experimental data, can find out, 6 pairs of primers for bunch design of Microcystin synthetic gene of the present invention, can get rid of false-positive generation by the mutual rectification of multipair primer, in addition, 6 pairs of primers have identical annealing temperature, can carry out pcr amplification simultaneously, improve widely amplification efficiency.

Claims

1. the primer sets of the PCR for detection of Microcystins in Water is characterized in that this PCR primer sets comprises following primer pair:

The primer pair of (1) 5 '-GATCCTCAGCAAMGDTTACT-3 ' and 5 '-GTTCCTGTGCCRTGAGCTTC-3 ';

The primer pair of (2) 5 '-AGAAATGACGCAACTGAATA-3 ' and 5 '-TGACCAAGTAATCCCAGAAC-3 ';

The primer pair of (3) 5 '-CTTACCCATTATTTACAGCA-3 ' and 5 '-CTTGAGTCATTTCGGGTTGG-3 ';

The primer pair of (4) 5 '-CAAGCCAAACTTTACCCTGAG-3 ' and 5 '-ATTCGGGACGATTGAGGTAT-3 ';

The primer pair of (5) 5 '-CCTACCGTCAATTAAACGAA-3 ' and 5 '-ATTACTAAACGGATTGGAGA-3 ';

The primer pair of (6) 5 '-ATCGCTATGCTGGCAGTATTT-3 ' and 5 '-CATTCAAAGGATTAGGCACA-3 '.