阅读说明：本技术 一种海水中海地瓜幼体生物量的检测统计方法 (Detection and statistics method for biomass of sea cucumber larvae in seawater ) 是由 蒋科技 刘守海 任远豪 夏利花 李磊 季晓 王亚美 申耀中 李子牛 宋炜 张凤英 于 2020-05-06 设计创作，主要内容包括：海水中海地瓜幼体生物量的检测统计方法包括：提取海地瓜成体DNA,测序得到COⅠ基因序列；以PCR产物为起始标准品制作4个浓度梯度的标准品；设计荧光定量PCR特异性引物并验证,通过后得到CT值-DNA浓度对数值的标准曲线；取不同干重海地瓜组织提取DNA,以不同干重的海地瓜组织的DNA为模板进行荧光定量PCR定量测试,得到DNA质量与海地瓜组织干重的趋势直线方程；测出待测海水样品中DNA的CT值,将CT值带入海地瓜组织干重-CT值推导关系式得到海地瓜幼体DNA的浓度值,计算得到所含海地瓜幼体的DNA质量；通过干湿重比例及推导关系式计算出海地瓜幼体的湿重。(The method for detecting and counting the biomass of the sea pachyrhizus larvae in the seawater comprises the following steps: extracting adult DNA of acaudina molpadioides, and sequencing to obtain a CO I gene sequence; preparing 4 concentration gradient standards by taking a PCR product as an initial standard; designing and verifying a fluorescent quantitative PCR specific primer, and obtaining a standard curve of CT value-DNA concentration logarithm value after the primer passes the verification; extracting DNA from acaudina molpadioides tissues with different dry weights, and performing fluorescence quantitative PCR quantitative test by taking the DNA of the acaudina molpadioides tissues with different dry weights as a template to obtain a trend linear equation of the DNA quality and the dry weight of the acaudina molpadioides tissues; measuring the CT value of the DNA in the seawater sample to be detected, substituting the CT value into a acaudina molpadioides tissue dry weight-CT value deduction relational expression to obtain a concentration value of the acaudina molpadioides larva DNA, and calculating to obtain the DNA quality of the contained acaudina molpadioides larva; and calculating the wet weight of the sea sweet potato larvae through the dry-wet-weight ratio and a derivation relational expression.)

1. A detection statistical method for biomass of sea cucumber larvae in seawater is characterized by comprising the following steps:

s1, extracting adult DNA of the acaudina molpadioides by using a marine animal genome DNA extraction kit, designing a primer of a CO I gene in the adult DNA of the acaudina molpadioides, performing PCR amplification by using the primer of the CO I gene, sequencing to obtain a CO I gene sequence, performing Blast comparison on the CO I gene sequence, and identifying that the CO I gene sequence is the CO I gene sequence of the acaudina molpadioides;

wherein, the primer sequence of the COI gene is as follows:

the upstream sequence: GACATGGCTTTCCCACGAATG

The downstream sequence: GCTCGTGTGTCTACGTCCA

When PCR amplification is carried out, the annealing temperature is 53 ℃;

s2, preparing 4 concentration gradients 10 using the PCR product obtained in step S1 as the starting standard^-2、10^-3、10^-4、10^-5The standard substance of (1);

s3, designing a fluorescent quantitative PCR specific primer according to the sequenced acaudina molpadioides COI gene sequence, verifying the specificity of the primer, and obtaining a standard curve of CT value-DNA concentration logarithm value and a corresponding linear equation through fluorescent quantitative PCR absolute quantification after the verification is passed;

the sequence of the specific primer of the fluorescent quantitative PCR is as follows:

the upstream sequence: GAGGTGTGGGTACAGGATGA

The downstream sequence: CTTAGTAAGAGGAGGAAGGCGG

During PCR amplification, the annealing temperature is as follows: 60 ℃;

s4, taking acaudina molpadioides tissues with different dry weights, extracting DNA, carrying out fluorescence quantitative PCR quantitative test by taking the DNA corresponding to the acaudina molpadioides tissues with different dry weights as a template, obtaining concentration values corresponding to the DNA of the acaudina molpadioides tissues with different dry weights according to the standard curve of the step S3, and further obtaining a trend linear equation of the DNA quality and the dry weight of the acaudina molpadioides tissues;

s5, combining the linear equation of the CT value-DNA concentration logarithm value in the step S3 and the linear equation of the DNA mass and the dry weight of the acaudina molpadioides tissue in the step S4 to obtain a derivation relational expression between the dry weight and the CT value of the acaudina molpadioides tissue, measuring the CT value of the DNA in the seawater sample to be detected through fluorescence quantitative PCR, substituting the CT value into the derivation relational expression to calculate the concentration value of the DNA of acaudina molpadioides larvae in the seawater sample to be detected, and calculating the DNA mass of the acaudina molpadioides larvae contained in the seawater sample to be detected according to the volume of the DNA in the seawater sample to be detected;

and S6, calculating the wet weight of the acaudina molpadioides larva through the dry-wet weight ratio of the acaudina molpadioides larva in the seawater sample to be detected and the derivation relational expression.

2. The method for statistical detection of the biomass of the larvae of the sweet potatoes in seawater as claimed in claim 1, wherein in step S3, the equation of the straight line of the standard curve of the CT value-DNA concentration logarithm value is: y-3.956 x + 13.139.

3. The method for detecting and counting the biomass of the acaudina molpadioides larvae in the seawater as claimed in claim 1, wherein in step S4, the different dry weights of the acaudina molpadioides tissue are respectively: 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 90mg, 120 mg.

4. The method according to claim 1, wherein in step S4, the equation of the trend of the DNA mass and the dry weight of the acaudina molpadioides tissue is: and y 0.2266 x.

5. The method according to claim 1, wherein in step S5, the dry weight of acaudina molpadioides tissue is derived from CT value as follows: n is a radical of_Measuring＝n_{Sign board}*10^(CT _Measuring ^{–13.139)/-3.956*1}*V_Measuring*10^-3/0.2266，n_Measuring＝n_{Sign board}*10^(CT _Measuring ^{–13.139)/-3.956*1}(N_MeasuringRepresents the test dry weight of acaudina molpadioides; n is_{Sign board}Indicating the concentration of the standard substance; CT_MeasuringRepresenting the CT value obtained by testing the water sample; v_MeasuringRepresenting the volume of a water sample DNA template; n is_MeasuringRepresenting the concentration of target DNA obtained by water sample test; n is_{Standard + test}And (4) representing the testing concentration of the standard substance after being mixed with the water sample.

6. The method for detecting and counting the biomass of the larvae of the acaudina molpadioides in the seawater as claimed in claim 1, wherein in step S6, the dry-wet weight ratio is: 26.2 percent.

Technical Field

The invention belongs to the field of biomass statistics of plankton, and particularly relates to a method for counting biomass of sea cucumber larvae in a sea water area of a nuclear power station.

Background

Pachyrhizus (Acaudina spp), belonging to Echinodermata (Echinodermata), Holothuroidea (Holothuroidea), Holothuroidea (Mypadida), Mylopanaceae (Caudidae), and Pectinathus (Acaudina). The genus has two species — pachyrhizus alba (Acaudina leucoprocta h.l.clark) and pachyrhizus hirta (Acaudina molpadioides Semper). Commonly known as sea eggplant, is abundant in subtropical sandy sea areas, Japan, Philippines and Indonesia, and is available in China at Hainan, Guangdong, Fujian and Zhejiang coastal areas. At present, researches on the acaudina molpadioides mainly focus on the aspects of nutritive value, medicinal value and physiological function of the acaudina molpadioides, and reports on biomass statistics of the acaudina molpadioides are few, and the research on Thangia molpadioides and the like shows that the acaudina molpadioides can cause the nuclear power station to block a water taking system in the sea area near the nuclear power station, so that the water taking safety is influenced, and certain threat effect exists on the operation safety of the nuclear power station. For the operation of the nuclear power station, the acaudina molpadioides is a disaster-causing organism, so that the estimation of the biomass of the acaudina molpadioides can effectively take measures to control the quantity of the acaudina molpadioides, thereby ensuring the healthy operation of the nuclear power station.

The current estimation method of plankton mainly comprises the following steps: cytometry, spectrophotometer, dry weight measurement, flow cytometry, and the like. At present, the biomass statistics of plankton mainly aims at a certain class, and an estimation method aiming at the biomass of a certain plankton in a sea area is relatively lacked according to the diameter size.

Disclosure of Invention

The invention provides a method for detecting and counting biomass of sea water middle-sea sweet potato larvae aiming at the problems and the defects in the prior art, solves the estimation problem of the biomass of the sea water middle-sea sweet potato larvae in a certain sea area, can estimate the biomass of the sea water sweet potato larvae in the certain sea area more quickly and accurately, and can provide reference for estimation of other aquatic organism larvae.

The invention solves the technical problems through the following technical scheme:

the invention provides a method for detecting and counting biomass of sea pachyrhizus larvae in seawater, which is characterized by comprising the following steps:

s1, extracting adult DNA of the acaudina molpadioides by using a marine animal genome DNA extraction kit, designing a primer of a CO I gene in the adult DNA of the acaudina molpadioides, performing PCR amplification by using the primer of the CO I gene, sequencing to obtain a CO I gene sequence, performing Blast comparison on the CO I gene sequence, and identifying that the CO I gene sequence is the CO I gene sequence of the acaudina molpadioides;

wherein, the primer sequence of the COI gene is as follows:

the upstream sequence: GACATGGCTTTCCCACGAATG

The downstream sequence: GCTCGTGTGTCTACGTCCA

When PCR amplification is carried out, the annealing temperature is 53 ℃;

s2, preparing 4 concentration gradients 10 using the PCR product obtained in step S1 as the starting standard^-2、10^-3、10^-4、10^-5The standard substance of (1);

s3, designing a fluorescent quantitative PCR specific primer according to the sequenced acaudina molpadioides COI gene sequence, verifying the specificity of the primer, and obtaining a standard curve of CT value-DNA concentration logarithm value and a corresponding linear equation through fluorescent quantitative PCR absolute quantification after the verification is passed;

the sequence of the specific primer of the fluorescent quantitative PCR is as follows:

the upstream sequence: GAGGTGTGGGTACAGGATGA

The downstream sequence: CTTAGTAAGAGGAGGAAGGCGG

During PCR amplification, the annealing temperature is as follows: 60 ℃;

s4, taking acaudina molpadioides tissues with different dry weights, extracting DNA, carrying out fluorescence quantitative PCR quantitative test by taking the DNA corresponding to the acaudina molpadioides tissues with different dry weights as a template, obtaining concentration values corresponding to the DNA of the acaudina molpadioides tissues with different dry weights according to the standard curve of the step S3, and further obtaining a standard curve of DNA quality and acaudina molpadioides tissue dry weight and a corresponding linear equation;

s5, combining a linear equation of the CT value-DNA concentration logarithm value in the step S3 and a linear equation of the trend between the DNA mass and the dry weight of the acaudina molpadioides tissue in the step S4 to obtain a derivation relational expression between the dry weight and the CT value of the acaudina molpadioides tissue, measuring the CT value of the DNA in the seawater sample to be detected through fluorescence quantitative PCR, substituting the CT value into the derivation relational expression to calculate the concentration value of the DNA of the acaudina molpadioides larva in the seawater sample to be detected, and calculating the DNA mass of the acaudina molpadioides larva contained in the seawater sample to be detected according to the volume of the DNA in the seawater sample to be detected;

and S6, calculating the wet weight of the acaudina molpadioides larva through the dry-wet weight ratio of the acaudina molpadioides larva in the seawater sample to be detected and the derivation relational expression.

Preferably, in step S3, the equation of the straight line of the standard curve of CT value-DNA concentration logarithm is: y-3.956 x + 13.139.

Preferably, in step S4, the different dry weights of the acaudina molpadioides tissue are: 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 90mg, 120 mg.

Preferably, in step S4, the equation of the straight line of the standard curve of DNA mass and acaudina molpadioides tissue dry weight is: and y 0.2266 x.

Preferably, in step S5, the derivation relationship between the dry weight of acaudina molpadioides tissue and the CT value is: represents the test dry weight of acaudina molpadioides; n is_{Sign board}Indicating the concentration of the standard substance; CT_MeasuringRepresenting the CT value obtained by testing the water sample; v_MeasuringRepresenting the volume of a water sample DNA template; n is_MeasuringRepresenting the concentration of target DNA obtained by water sample test; n is_{Standard + test}And (4) representing the testing concentration of the standard substance after being mixed with the water sample.

Preferably, in step S6, the dry-wet weight ratio is: 26.2 percent.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The positive progress effects of the invention are as follows:

the invention provides a method for rapidly detecting biomass of acaudina molpadioides in a seawater area, which can estimate the outbreak period of the acaudina molpadioides biomass through testing, thereby providing certain time for taking preventive measures and achieving outbreak early warning and disaster control of the acaudina molpadioides biomass.

According to the method, DNA is extracted from a water sample in the sea area of the nuclear power station, a specific primer is designed to perform a fluorescence quantitative PCR experiment to obtain a CT value, and the CT value obtained through testing is directly introduced into a derivation formula, so that the biomass of the sea cucumber larvae in a certain sea area can be directly, accurately, efficiently and quickly estimated, the defects of high workload and high error of a traditional estimation method are overcome, the time and material cost is greatly reduced, the workload is reduced, the outbreak threshold and the outbreak period of the sea cucumber biomass in the sea area of the nuclear power station can be predicted, a certain early warning and precautionary measure preparation and a certain technical reference for the biomass estimation of other marine organism larvae are provided for the management and control of the sea cucumber biomass in the sea area of the nuclear power station, and the method has a good application prospect.

Drawings

FIG. 1 is a schematic diagram of the verification of primer specificity according to a preferred embodiment of the present invention, wherein a: DNA mixtures of other species; b: other species DNA mixture + acaudina molpadioides DNA template; c: DNA template of the acaudina molpadioides; d: water sample environment DNA.

FIG. 2 is a schematic diagram of a melting curve according to a preferred embodiment of the present invention.

FIG. 3 is a diagram illustrating a standard curve of CT value versus DNA concentration log value according to a preferred embodiment of the present invention.

FIG. 4 is a graph showing the standard curve of DNA quality and tissue dry weight of acaudina molpadioides according to the preferred embodiment of the present invention.

FIG. 5 is a schematic diagram of an amplification curve of a test water sample according to a preferred embodiment of the present invention.

FIG. 6 is a schematic diagram of the amplification curve of the test water sample + the target DNA according to the preferred embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The embodiment provides a method for detecting and counting biomass of sea cucumber larvae in seawater, which comprises the following steps:

s1, extracting adult DNA of the acaudina molpadioides by using a marine animal genome DNA extraction kit, designing a primer of a CO I gene in the adult DNA of the acaudina molpadioides, performing PCR (polymerase chain reaction) amplification by using the primer of the CO I gene, performing sequencing to obtain a CO I gene sequence when the annealing temperature of the PCR amplification is 53 ℃, and performing Blast comparison on the CO I gene sequence to identify that the CO I gene sequence is the CO I gene sequence of the acaudina molpadioides.

Wherein, the primer sequence and PCR preparation of the COI gene are respectively shown in tables 1-1 and 1-2:

TABLE 1-1 Pachyrhizus Erosi COI gene PCR primers

TABLE 1-2 general PCR preparation of 25. mu.L System

S2, preparing 4 concentration gradients 10 by using the PCR product obtained in the step S1 and the purified adult DNA of the acaudina molpadioides as initial standard products^-2、10^-3、10^-4、10^-5The standard substance of (1).

S3, designing a fluorescent quantitative PCR specific primer according to the sequenced acaudina molpadioides COI gene sequence, and verifying the specificity of the primer (as shown in figure 1 and figure 2), wherein during PCR amplification, the annealing temperature is as follows: and (3) after the verification is passed, obtaining a standard curve of CT value-DNA concentration logarithm value and a corresponding linear equation by fluorescence quantitative PCR absolute quantification: y is-3.956 x +13.139 and the standard curve is shown in figure 3.

The specific primer sequences and reaction systems of the fluorescent quantitative PCR are shown in tables 2-1 and 2-2 respectively:

TABLE 2-1 fluorescent quantitative PCR specific primers for acaudina molpadioides

TABLE 2-2 fluorescent quantitative PCR reaction System

S4, taking acaudina molpadioides tissues with different dry weights, extracting DNA, carrying out fluorescence quantitative PCR quantitative test by taking the DNA corresponding to the acaudina molpadioides tissues with different dry weights as a template, obtaining concentration values corresponding to the DNA of the acaudina molpadioides tissues with different dry weights according to the standard curve of the step S3, and further obtaining a standard curve of DNA mass (mug) and acaudina molpadioides tissue dry weight (mg) and a corresponding linear equation: y 0.2266x, the standard curve is as in fig. 4.

Wherein, the different dry weights of acaudina molpadioides tissue are respectively: 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 90mg, 120 mg.

S5, combining the linear equation of the CT value-DNA concentration logarithm value in the step S3 and the linear equation of the trend between the DNA mass and the dry weight of the acaudina molpadioides tissue in the step S4 to obtain a derivation relational expression between the dry weight and the CT value of the acaudina molpadioides tissue, measuring the CT value of the DNA in the seawater sample to be detected through fluorescence quantitative PCR, substituting the CT value into the derivation relational expression to calculate the concentration value of the DNA of the acaudina molpadioides larva in the seawater sample to be detected, and calculating the DNA mass (mu g) of the acaudina molpadioides larva contained in the seawater sample to be detected according to the volume of the DNA in the seawater sample to be detected.

Wherein, the derivation relation between the dry weight of the acaudina molpadioides tissue and the CT value is as follows:

N_measuringRepresents the test dry weight of acaudina molpadioides; n is_{Sign board}Indicating the concentration of the standard substance; CT_MeasuringRepresenting test water samplesThe obtained CT value; v_MeasuringRepresenting the volume of a water sample DNA template; n is_MeasuringRepresenting the concentration of target DNA obtained by water sample test; n is_{Standard + test}And (4) representing the testing concentration of the standard substance after being mixed with the water sample.

And S6, calculating the wet weight of the acaudina molpadioides larva through the dry-wet weight proportion (26.2%) of the acaudina molpadioides larva in the seawater sample to be detected and a derivation relational expression.

Taking Fujian Qingchuan bay sea area as an example, a water sample is taken, DNA is extracted by using a marine animal tissue genome DNA extraction kit-centrifugal column type, and the extracted DNA is stored at the temperature of minus 20 ℃. Then, the fluorescence quantitative PCR experiment was performed, the system is shown in Table 2-2, the results are shown in FIG. 5, the CT value is 23.505, and the results are respectively substituted into the formulas Obtaining: n is_Measuring＝1.781*10^-1ng/μL，N_Measuring＝1.572*10^-3mg。

The verification method comprises the following steps: adding an isovolumetric standard substance into a template for testing the DNA of a water sample, observing the change condition of the CT value, and according to the formula And performing calculation verification. (wherein N is_Testing: dry weight of the acaudina molpadioides in a water sample; n is_{Standard + test}(ii) a The mixed concentration after adding the standard substance; CT_Measuring: testing a CT value by using a water sample; CT_{Standard + test}: mixing the water sample DNA with a standard substance and then testing a corresponding CT value for the template; v_Measuring: the volume of the DNA template of the test water sample is 2 mu L)

After the equal volume of the standard substance is added, the change of the CT value is shown in figure 6, and the amplification map (CT value: 21.040) and the amplification map (CT value: 23.505) of the water sample and the standard substance after the water sample and the standard substance are mixed are delta CT_{Authentication}23.505-21.040-2.465. Therefore, the method comprises the following steps: n is_Measuring/n_{Survey + mark}＝10^{ΔCT/-3.956*1}Substituting to obtain: n is_Measuring＝10.0444ng/μL，N_Measuring＝8.865*10^-2And (5) mg. The error is Δ N/N × 100% ═ 55.393(Δ N is the difference between the verified target DNA concentration value of the water sample and the actually measured target DNA concentration value of the water sample), that is: about 55.393%.

While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that these are by way of example only, and that the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications are within the scope of the invention.