CN102242191A

CN102242191A - PCR (polymerase chain reaction) primer for detecting microcystins in water bodies and application thereof

Info

Publication number: CN102242191A
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: 2011-11-16
Anticipated expiration: 2031-04-25
Also published as: CN102242191B

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

Be used for detecting the PCR primer and the application thereof of water body Microcystin

Technical field

The present invention relates to a kind of PCR primer that is used for detecting the water body Microcystin, the invention still further relates to this PCR primer detects Microcystin in water body application.

Background technology

Blue-green algae has another name called " blue-green algae ", and in some nutritious water bodys, often a large amount of breedings form " wawter bloom " to some blue-green algae summer.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.Cyanophycean toxin can be divided into four big classes according to the difference of its illness that finally causes: 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 eliminating cyanophycean toxin, the scientific worker of various countries has carried out number of research projects.

For the detection of Microcystin, carry out the bio-toxicity experiment whether to have cyanophycean toxin to exist in the detection water body by making up corresponding living model in early days.Yet a series of ethics problems that lower sensitivity, higher experiment expend and the bio-toxicity experiment brings make people have to select method for distinguishing to come cyanophycean toxin is diagnosed.Along with the micro-example analytical instrument (as, HPLC, MALDI-TOF) development, rely on these instrument developments to go out the multiple method that is used for detecting the natural water cyanophycean toxin.These methods analyst process simple and fasts and have 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 the physical environment water body gradually.As, ELISA method, colorimetry (protein phosphatase inhibitor) and cytotoxicity that hepatotoxin (microcystin and nodularin) detects are diagnosed.

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

Summary of the invention

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

In the biological synthesis gene cluster of Microcystin, mcyA, mcyB, mcyC, mcyD, these 6 main big fragment genes of mcyE, mcyG have very important effect to Microcystin synthetic.On the other hand, the malicious Microcystis aeruginosa of non-product also may contain part product virus gene.Therefore, we bunch have designed 6 pairs of primers at the Microcystin synthetic gene.To get rid of false-positive generation by many mutual rectifications to primer, also make 6 pairs of primers have identical annealing temperature simultaneously, can carry out pcr amplification simultaneously, improve amplification efficiency.

The primer design theory: at 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; At the A structural domain design synthetic primer of the D-MeAsp of mcyB genes encoding to MCYBAF MCYBAR; At 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; At 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 that by the specificity analysis and assessment it is consistent with the LC-MS detected result to use the Microcystin detected result that 6 pairs of primers carry out, 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 because single primer is detected forms false-positive phenomenon.In sum, with respect to the detection of Microcystin traditional in the water body, use these 6 pairs of primers of sophisticated round pcr can be quick, sensitive detects in the water body whether have Microcystin, thereby improves sensitivity and the efficient that detects greatly.

Description of drawings

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

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

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

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

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

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

Embodiment

Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.

Embodiment:

1, algal species cultivation: buy ten strain algae kinds from the typical case culture collection council of Chinese Academy of Sciences algae kind storehouse (FACHB Collection), 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 are 1000-1500lux, and Light To Dark Ratio is 12: 12, shake every day bottle 1-2 time, leave standstill 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: MCYST is microcystin in the table, and " no " expression does not detect toxin

3, the PCR of toxin synthetic gene bunch detects: at the primer of Microcystin synthetic gene bunch design ten strain Microcystis aeruginosas are carried out pcr amplification with 6 couple of our design.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 Fig. 1～shown in Figure 6, the PCR band is special and single.M:Marker2000 molecular weight standard among 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 among 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: "-": ecbatic is negative, "+": be expressed as the positive.

From above experimental data as can be seen, 6 pairs of primers at bunch design of Microcystin synthetic gene of the present invention, can get rid of false-positive generation by many mutual rectifications to primer, in addition, 6 pairs of primers have identical annealing temperature, can carry out pcr amplification simultaneously, improve amplification efficiency widely.

Claims

1. PCR primer that is used for detecting the water body Microcystin is characterized in that this PCR primer is selected from following each primer sets:

(1) comprises primer with 5 '-GATCCTCAGCAAMGDTTACT-3 ' and have the primer sets of the primer of 5 '-GTTCCTGTGCCRTGAGCTTC-3 ';

(2) comprise primer with 5 '-AGAAATGACGCAACTGAATA-3 ' and have the primer sets of the primer of 5 '-TGACCAAGTAATCCCAGAAC-3 ';

(3) comprise primer with 5 '-CTTACCCATTATTTACAGCA-3 ' and have the primer sets of the primer of 5 '-CTTGAGTCATTTCGGGTTGG-3 ';

(4) comprise primer with 5 '-CAAGCCAAACTTTACCCTGAG-3 ' and have the primer sets of the primer of 5 '-ATTCGGGACGATTGAGGTAT-3 ';

(5) comprise primer with 5 '-CCTACCGTCAATTAAACGAA-3 ' and have the primer sets of the primer of 5 '-ATTACTAAACGGATTGGAGA-3 ';

(6) comprise primer with 5 '-ATCGCTATGCTGGCAGTATTT-3 ' and have the primer sets of the primer of 5 '-CATTCAAAGGATTAGGCACA-3 '.

2. the described PCR primer of claim 1 application in the Microcystin in detecting water body.