qPCR和熒光光譜：有毒藍(lán)藻實(shí)時(shí)定量監(jiān)測(cè)技術(shù)的應(yīng)用分析

藻類(lèi)暴發(fā)可由營(yíng)養(yǎng)物、溫度、光照、水文條件等環(huán)境因素以及電導(dǎo)率、鹽度、濁度等化學(xué)指標(biāo)的變化引起。由于全球氣候的變化，藻類(lèi)暴發(fā)事件的數(shù)量在北美(五大湖地區(qū))及亞洲眾多地區(qū)(中國(guó)、東南亞等)正以驚人的速度增漲[1]。藍(lán)藻是藻類(lèi)暴發(fā)的主導(dǎo)物種，會(huì)產(chǎn)生藻毒素等致命產(chǎn)物，對(duì)人類(lèi)和水生生態(tài)系統(tǒng)造成危害；其中微囊藻毒素不僅會(huì)損傷人體的肝臟和神經(jīng)系統(tǒng)，同時(shí)也是一種潛在的致癌物質(zhì)[2]。目前，關(guān)于海洋、咸水及淡水中有毒藍(lán)藻鑒定和監(jiān)測(cè)的文獻(xiàn)研究并不鮮見(jiàn)，然而對(duì)于穩(wěn)定可靠的24/7藻類(lèi)實(shí)時(shí)監(jiān)測(cè)系統(tǒng)的需求卻迫在眉睫。實(shí)時(shí)監(jiān)測(cè)系統(tǒng)須能在藻類(lèi)暴發(fā)前后實(shí)現(xiàn)有毒藍(lán)藻的快速檢測(cè)和有效鑒定，以防止魚(yú)蝦大規(guī)模死亡，確保供水安全。

自20世紀(jì)60年代初期開(kāi)始，熒光檢測(cè)技術(shù)逐漸開(kāi)始受到關(guān)注。當(dāng)光被葉綠素分子吸收時(shí)，其中一部分吸收的能量會(huì)以熒光的形式重新輻射出來(lái)，而熒光檢測(cè)技術(shù)能通過(guò)識(shí)別熒光輻射產(chǎn)生的特征激發(fā)光譜來(lái)分析光合細(xì)菌(如藍(lán)藻)中的葉綠素分子，省時(shí)省力，并能收集大量的監(jiān)測(cè)數(shù)據(jù)。

與此同時(shí)，近十年來(lái)，定量聚合酶鏈反應(yīng)(quantitative polymerase chain reaction，qPCR)技術(shù)逐漸得到應(yīng)用，即通過(guò)采用實(shí)時(shí)聚合酶鏈反應(yīng)的分子探針來(lái)鑒別水環(huán)境系統(tǒng)中特定的藍(lán)藻種類(lèi)。這一快速便捷的實(shí)驗(yàn)室技術(shù)目前正在迅速發(fā)展，以實(shí)現(xiàn)實(shí)時(shí)的24/7藻類(lèi)監(jiān)測(cè)。

本文主要對(duì)比了傳統(tǒng)的光譜分析法和迅速發(fā)展的分子學(xué)方法——qPCR技術(shù)在藍(lán)藻監(jiān)測(cè)方面的應(yīng)用。首先簡(jiǎn)要介紹了兩種方法的背景，隨后對(duì)兩者的優(yōu)勢(shì)進(jìn)行比較。

1　傳統(tǒng)熒光光譜分析法

葉綠素-a是一種光合色素，存在于包括真核生物(藻類(lèi))和原核生物(藍(lán)藻)在內(nèi)的所有浮游植物中，是用于計(jì)量浮游植物總生物質(zhì)的有效且廣泛使用的指標(biāo)。然而藍(lán)藻的色素系統(tǒng)產(chǎn)生的葉綠素-a熒光信號(hào)非常微弱。與此同時(shí)，藍(lán)藻的熒光發(fā)生系統(tǒng)主要位于光系統(tǒng)II，而光系統(tǒng)II主要由藻青蛋白(PC)和藻紅蛋白(PE)等藻膽色素構(gòu)成[3-4]。因此可通過(guò)分析藻膽色素?zé)晒夤庾V攜帶的大量信息，測(cè)算藍(lán)藻的豐度，這就需要對(duì)藻膽色素的熒光光譜進(jìn)行優(yōu)化。

圖1顯示了四種不同的藻類(lèi)和黃色物質(zhì)(yellow substances,YS)的標(biāo)準(zhǔn)光譜[5]。對(duì)于藍(lán)藻色素來(lái)說(shuō)，PC所在的藍(lán)藻組和PE所在的紅藻組分別在610 nm和570 nm的波長(zhǎng)下檢測(cè)到了強(qiáng)烈的熒光信號(hào)，因此可通過(guò)最優(yōu)激發(fā)波長(zhǎng)的改變，實(shí)現(xiàn)某一特定藻類(lèi)熒光標(biāo)記的優(yōu)化?；诖?，在680 nm的發(fā)射波長(zhǎng)下，采用發(fā)光二極管，microLAN系統(tǒng)[6]可分別在570、615 nm和450 nm的選定激發(fā)波長(zhǎng)下檢測(cè)PE、PC和葉綠素-a產(chǎn)生的熒光強(qiáng)度。此外，對(duì)于水樣中一些會(huì)干擾檢測(cè)的其他熒光物質(zhì)，如溶解性有機(jī)物(365 nm)、濁度(710 nm)等，系統(tǒng)也會(huì)進(jìn)行相應(yīng)的修正。

圖1　四種不同藻類(lèi)和黃色物質(zhì)的標(biāo)準(zhǔn)光譜圖

2　分子探針——qPCR技術(shù)

自人類(lèi)基因組計(jì)劃(1990年～2003年)開(kāi)始以來(lái)，快速分子生物學(xué)工具的開(kāi)發(fā)應(yīng)用得到了大量關(guān)注，它們可用來(lái)鑒定存在于敏感生態(tài)系統(tǒng)中且對(duì)人類(lèi)具有毒性的特定微生物種類(lèi)[7]。首次采用實(shí)時(shí)PCR技術(shù)來(lái)監(jiān)測(cè)有毒的浮游植物是在2000年。藍(lán)藻專(zhuān)有的基因標(biāo)記可以以負(fù)責(zé)生成微囊藻毒素、節(jié)球藻毒素、蛤蚌毒素以及擬柱孢藻毒素等毒素的產(chǎn)毒基因?yàn)槟繕?biāo)。

Koskeenniemi等[8]的研究采用特定的PCR引物(SYBR green)，同時(shí)以結(jié)球藻毒素基因亞基(nDaF)為目標(biāo)，結(jié)果表明，波羅的海水樣中的基因拷貝數(shù)(即藍(lán)藻數(shù)量)和結(jié)球藻毒素的濃度存在強(qiáng)烈的相關(guān)性。該qPCR技術(shù)可用于具體研究波羅的海中結(jié)球藻的暴發(fā)和結(jié)球藻毒素的形成。

一篇研究報(bào)告在綜述33項(xiàng)研究(2003年～2015年)的基礎(chǔ)上，通過(guò)分析微囊藻毒素濃度和qPCR數(shù)據(jù)之間的相關(guān)性，對(duì)于qPCR技術(shù)能否可靠指示淡水中的藍(lán)藻毒素風(fēng)險(xiǎn)進(jìn)行了評(píng)述[9]。根據(jù)相關(guān)性分析，其中的22項(xiàng)研究顯示為正相關(guān)(65%)，11項(xiàng)為無(wú)相關(guān)性(33%)。然而，對(duì)于上述相同的研究組來(lái)說(shuō)，在檢驗(yàn)微囊藻毒素濃度和葉綠素-a或藍(lán)藻細(xì)胞數(shù)量之間的相關(guān)性時(shí)，85%的情況顯示為正相關(guān)。因此，通過(guò)與傳統(tǒng)方法的比較，研究者對(duì)于采用qPCR技術(shù)來(lái)評(píng)估有毒藍(lán)藻暴發(fā)風(fēng)險(xiǎn)的可靠性提出了質(zhì)疑。

盡管采用qPCR技術(shù)來(lái)評(píng)估藍(lán)藻暴發(fā)毒性，其結(jié)果有時(shí)存在爭(zhēng)議，但qPCR技術(shù)在藍(lán)藻菌群的動(dòng)力學(xué)研究中仍具有重要價(jià)值，比如研究有毒菌株相對(duì)豐度的時(shí)間和空間變化、分析藍(lán)藻暴發(fā)的動(dòng)力學(xué)情況及其與環(huán)境因素之間的關(guān)系等[10-11]。qPCR技術(shù)的另一大優(yōu)勢(shì)是它能同時(shí)檢測(cè)多個(gè)目標(biāo)基因，稱(chēng)為“多重qPCR”(multiplex qPCR)，比如可同時(shí)將微囊藻毒素、節(jié)球藻毒素、擬柱孢藻毒素以及蛤蚌毒素四種有毒的生物合成基因簇作為目標(biāo)。對(duì)51種毒性和非毒性藍(lán)藻菌株的檢測(cè)已證實(shí)了多重qPCR技術(shù)的特異性。采用該方法，成功檢測(cè)了澳大利亞墨累河中混合藍(lán)藻水華的毒性[12]。

3　方法比較

采用兩種技術(shù)手段，對(duì)現(xiàn)場(chǎng)條件下的藍(lán)藻進(jìn)行24/7實(shí)時(shí)監(jiān)測(cè)，并對(duì)兩種方法進(jìn)行了比較。需要指出的是，盡管實(shí)時(shí)qPCR技術(shù)已有實(shí)驗(yàn)室現(xiàn)場(chǎng)原型(field prototype)，但目前還沒(méi)有可商用的qPCR系統(tǒng)來(lái)實(shí)現(xiàn)對(duì)藍(lán)藻的24/7現(xiàn)場(chǎng)實(shí)時(shí)監(jiān)測(cè)。然而，qPCR技術(shù)目前正在迅速發(fā)展，且其在藍(lán)藻暴發(fā)的前期監(jiān)測(cè)中具有關(guān)鍵作用，因此有必要對(duì)兩種方法進(jìn)行比較。

表1總結(jié)了兩種方法的關(guān)鍵差別。傳統(tǒng)光譜方法通過(guò)識(shí)別不同光合色素(葉綠素-a、藻青蛋白、藻紅蛋白等)特有的激發(fā)-發(fā)射光譜特征來(lái)鑒定不同的藻類(lèi)，而qPCR技術(shù)則通過(guò)先進(jìn)的分子學(xué)手段鑒別出不同種類(lèi)的藍(lán)藻。由于qPCR技術(shù)在樣品的運(yùn)送和準(zhǔn)備過(guò)程中都需要專(zhuān)業(yè)生物測(cè)定手段的支持，因此參與qPCR實(shí)時(shí)監(jiān)測(cè)的技術(shù)人員所需具備的專(zhuān)業(yè)門(mén)檻要高于采用傳統(tǒng)方法的技術(shù)人員。如果是在發(fā)展中國(guó)家或偏遠(yuǎn)地區(qū)應(yīng)用qPCR技術(shù)，這類(lèi)在分子生物學(xué)領(lǐng)域具備專(zhuān)業(yè)能力的技術(shù)人員將更難招募。

基于傳統(tǒng)光譜法的24/7實(shí)時(shí)監(jiān)測(cè)系統(tǒng)通常是以探針或固定化系統(tǒng)的形式在現(xiàn)場(chǎng)部署，市面上容易購(gòu)得相關(guān)設(shè)備，其成本一般由監(jiān)測(cè)范圍的大小而定，通常可負(fù)擔(dān)得起。因此相對(duì)而言，基于qPCR技術(shù)的24/7實(shí)時(shí)監(jiān)測(cè)系統(tǒng)成本可能要高得多。

表1　傳統(tǒng)光譜技術(shù)和qPCR技術(shù)的比較

在未來(lái)3～5年內(nèi)，我們有望看到傳統(tǒng)方法向商用24/7實(shí)時(shí)流式細(xì)胞監(jiān)測(cè)法(flow cytometry monitoring)的發(fā)展進(jìn)化[13]；而對(duì)于qPCR技術(shù)來(lái)說(shuō)，基于“芯片實(shí)驗(yàn)室”(lab-on-a-chip)技術(shù)的現(xiàn)場(chǎng)原型將使得實(shí)時(shí)監(jiān)測(cè)系統(tǒng)的商業(yè)化應(yīng)用更具可行性[14-15]。

4　結(jié)論

采用熒光光譜技術(shù)鑒定藍(lán)藻是一項(xiàng)突破性的應(yīng)用，是實(shí)現(xiàn)快速、可靠、24/7實(shí)時(shí)的水系統(tǒng)藍(lán)藻暴發(fā)監(jiān)測(cè)的有力手段。目前傳統(tǒng)的光譜學(xué)方法已廣為人知，且在24/7藻類(lèi)實(shí)時(shí)監(jiān)測(cè)系統(tǒng)中廣泛應(yīng)用，與此同時(shí)采用分子學(xué)技術(shù)的qPCR方法在過(guò)去十年間也得到了迅速發(fā)展。qPCR方法能夠單獨(dú)鑒定一種或同時(shí)鑒定多種(4～5種)有毒藍(lán)藻菌種，具有顯著優(yōu)勢(shì)。在全球氣候環(huán)境越來(lái)越多變的狀況下，傳統(tǒng)的光譜技術(shù)和先進(jìn)的qPCR技術(shù)將繼續(xù)共同發(fā)揮關(guān)鍵作用，為研究人員、監(jiān)管人員和決策人員在藻類(lèi)暴發(fā)的管理和防治方面提供重要信息和依據(jù)。

[1]Wilhelm S W,Farnsley S E,LeCleir G R,etal.The relationships between nutrients,cyanobacterial toxins and the microbial community in Taihu (Lake Tai),China[J].Harmful Algae,2011,10(2):207-215.

[2]USGS.The science of harmful algalblooms[EB/OL].[2016-10-24].https://www.usgs.gov/news/science-harmful-algae-blooms.

[3]Sobiechowska Sasim M,Stoń Egiert J,Kosakowska A.Quantitative analysis of extracted phycobilin pigments in cyanobacteria-An assessment of spectrophotometric and spectrofluorometric methods[J].Journal of Applied Phycology,2014,26(5):2065-2074.

[4]Simis S G H,Huot Y,Babin M,etal.Optimization of variable fluorescence measurements of phytoplankton communities with cyanobacteria[J].Photosynthesis Research,2012,112(1):13-30.

[5]Beutler M.Spectral fluorescence of chlorophyll and phycobilins as an in-situ tool of phytoplankton analysis models,algorithms and instruments[D].Kiel,Germany:Christian-Albrechts-Universit?t zu Kiel,2003.

[6]MicroLan.ALGControl:Fluorescence monitoring of algae classes and toxic algae[EB/OL].http://www.microlan.nl/monitoring-products/algcontrol-fluorescence-monitoring-algae/.

[7]Genome News Network.Sequencing the genome of Haemophilus influenza Rd[EB/OL].http://www.genomenewsnetwork.org/resources/timeline/1995_Haemophilus.php.

[8]Koskenniemi K,Lyra C,Rajaniemi-Wacklin P,etal.Quantitative real-time PCR detection of toxic Nodularia cyanobacteria in the Baltic Sea[J].Applied and Environmental Microbiology,2007,73(7):2173-2179.

[9]Pacheco A B F,Guedes I A,Azevedo S M F O.Is qPCR a reliable indicator of cyanotoxin risk in freshwater[J].Toxins,2016,8(6):172-172.

[10]Martins A,Vasconcelos V.Use of qPCR for the study of hepatotoxic cyanobacteria population dynamics[J].Archives of Microbiology,2011,193(9):615-615.

[11]Antonella P,Luca G.The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB)species[J].Environmental Science and Pollution Research,2013,20(10):6851-6862.

[12]Al-Tebrineh J,Merrick C,Ryan D,etal.Community composition,toxigenicity,and environmental conditions during a cyanobacterial bloom occurring along 1 100 kilometers of the Murray River[J].Applied and Environmental Microbiology,2012,78(1):263-272.

[13]Besmer M D,Weissbrodt D G,Kratochvil B E,etal.The feasibility of automated online flow cytometry for in-situ monitoring of microbial dynamics in aquatic ecosystems[J].Frontiers in Microbiology,2014,5(265):265-265.

[14]Weller M G.Immunoassays and biosensors for the detection of cyanobacterial toxins in water[J].Sensors,2013,13(11):15085-15112.

[15]Courtois S,Jary D,Do-Quang Z,etal.Developing a fully-integrated microdevice for the in-situ detection of cyanobacteria and cyanotoxin-producing strains in fresh water samples[C].Busan,Korea:Proceedings of the IWA World Water Congress,2012.

【原文】

UtilizingAdvancedFluorescenceTechnologyforReal-TimeMonitoringofToxicCyanobacteriainAlgalBlooms

CharlesCCLee1,JoepAppels2,ZhangXianchao3

(1.University of Newcastle,Singapore; 2.MicroLAN,Netherlands; 3.Zean Inc,China)

Introduction

Algal blooms arelikely caused by environmental factors such as:nutrients; temperature; light; hydrology; and water chemistry (e.g.,pH,conductivity,salinity,turbidity).Due to climate change,algal blooms are increasing at an alarming rate across North America (Great Lakes) and numerous regions in Asia (China,Southeast Asia)[1].Cyanobacteria is the dominant species in algal blooms which produces lethal cyanotoxins harmful to humans and the aquatic ecosystem.Microcystin toxins damages the liver and the nervous system,as well as a potent carcinogen[2].While there is no lack of literature on the identification and monitoring of toxic cyanobacteria in marine,brackish and freshwaters,there is an urgent need for a reliable real-time 24/7 algal monitoring system.This system would allow for a quick detection of toxic cyanobacteria before,during and after algal blooms in order to protect water supplies and prevent massive fish kills.

The analysis of chlorophyll molecules in photosynthetic bacteria (e.g.cyanobacteria) by identification of the unique excitation spectra produced from fluorescence has gained significant interest since the early 1960s.Fluorescence monitoring is extremely attractive as it is quick,not labor intensive,and enables the collection of massive amount of monitoring data.When chlorophyll molecules absorb light,a fraction of the energy absorbed is re-emitted as fluorescence.More recently,in the last decade,molecular probes using a real-time polymerase chain reaction (PCR),also known as quantitative PCR (qPCR) technology were developed to identify specific cyanobacteria species in water systems.This is currently a quick laboratory-based technique that is being rapidly developed experimentally for real-time 24/7 monitoring in the field.

The focusof this paper is to compare the monitoring of cyanobacteria using the traditional spectroscopic method with the rapidly advanced molecular method-qPCR.First a brief background of both methods will be described,followed by a comparison of the advantages of both methods.

TraditionalSpectroscopicMethod

Chlorophyll-a is a photosynthetic pigment present in all species of phytoplankton,including eukaryotic (algae) and prokaryotic organisms (cyanobacteria) thus it is a reliable and commonly used proxy for total phytoplankton biomass.However,the pigment system of cyanobacteria produces only a weak chlorophyll-a fluorescence signal.The cyanobacteria′s fluorescence system arise mainly from photosystem II.Photosystem II is comprised of pigments called phycobilins - e.g.phycocyanin (PC) and phycoerythrin (PE)[3-4].Therefore,it is important to optimize the fluorescence spectra of phycobilins,carrying a significant amount of spectral information that can be used to assess the abundance of cyanobacteria.

Figure 1 shows the normal spectra of four algal groups and yellow substances (YS)[5].Focusing on the cyanobacteria pigments,a strong signal is found for PC at 610 nm in the “blue group”.For PE,a strong signal is detected at 570 nm in the “red group” (red algae).It is therefore,possible to optimize the fluorescence signature of specific algae by switching on the optimal excitation wavelength.Using light emitting diodes (LED) at selected excitation wavelengths with an emission wavelength of 680nm,the microLan system[6]detects PE,PC and chlorophyll-a at 570,615 and 450 nm,respectively.Additionally,correction can be applied for other fluorescing matters such as dissolved organic matter (365 nm),and turbidity (710 nm) in the water sample that interferes with the measurement.

MolecularProbe-qPCRMethod

Since the discovery of the human genome project (1990-2003) there has been an explosive interest in developing rapid molecular biology tools to identify specific microbial species that are toxic to humans and present in sensitive ecosystems[7].The first applications of real-time PCR to monitor toxic phytoplankton was conducted in the year 2000.Genetic markers,unique to cyanobacteria,target toxin producing genes that are responsible for the synthesis of toxins.These toxins include microcystins,nodularin,saxitoxin,and cylindrospermopsin.

Using specific PCR primers (SYBR green) targeting the nodularin gene subunit (nDaF),Koskeenniemietal(2007)[8]showed a strong correlation between gene copy numbers (quantity of cyanobacteria) and nodularin concentrations in Baltic seawater samples.This qPCR technique can be used for detailed studies of Nodularin blooms and formation in the Baltic sea.

An extensive research paper reviewing 33 studies (2003-2015) on whether qPCR is a reliable indicator of cyanotoxin risk in freshwater correlated qPCR data with microcystin concentrations[9].The correlation analysis showed the following results:positive correlation for 22 studies (65%),and no correlation for 11 studies (33%).However,for the same set of studies above,when the correlation between the microcystin concentration and chlorophyll-a or number of cyanobacterial cells was tested,it was positive in 85% of the cases.The reviewer therefore questioned the reliability of using qPCR in comparison with traditional methods for the risk assessment of toxic cyanobacterial blooms.

Although the use of qPCR is sometimes questioned for estimating the toxicityof cyanobacterial blooms,it is still considered valuable for the study of cyanobacteria population dynamics.This includes exploring temporal and spatial variations in the relative abundance of toxic strains,and understanding of the dynamics of cyanobacterial blooms and their relationships with environmental factors[10-11].An additional advantage of qPCR is the ability to detect more than one single target gene called multiplex qPCR.Multiplex qPCR therefore can target four toxin biosynthesis gene clusters simultaneously such as microcystin,nodularin,cylindrospermopsin,and saxitoxin.The specificity of the multiplex qPCR was validated by testing 51 toxic and non-toxic cyanobacterial strains.Using this method,the toxigenicity of mixed cyanobacterial blooms in the Murray River (Australia) was successfully tested[12].

Comparisons

The comparison of the two methods is focused on implementation of the technology for 24/7 monitoring of cyanobacteria under real-time field conditions.To our knowledge,whilethere are experimental field prototypes of real-time qPCR,there is currently no commercially available qPCR system deployed to conduct 24/7 cyanobacteria monitoring in the field.However,the qPCR is rapidly advancing and certainly plays a crucial role in the early monitoring of cyanobacteria blooms.Therefore,it is worthwhile to compare the two methods.

Table 1 summarises the key differences between the two methods.While the traditional spectroscopic technology focuses on identification using the unique excitation-emission signature of photosynthetic pigments (chlorophyll-a,phycocyanin,phycoerthrin),qPCR specifically identifies the cyanobacteria species based on advanced molecular techniques.Because the qPCR requires specialized biological assays in both delivery and preparation,the skill sets of the technicians involved in conducting the monitoring would be significantly higher than those required to deploy the traditional method.This specialized skill sets in molecular biology is likely more difficult to find if the qPCR technology is to be deployed in developing economies or in remote areas.

Commercially available 24/7 monitoring systems for cyanobacteria utilizing the traditional spectroscopic method can be readily purchased on the market as deployable probes and stationary systems.Depending on the scale of the implementation,costs for such systems are generally affordable.In comparison,it is likely to be significantly more costly to develop a real-time 24/7 qPCR monitoring system.

Tab.1　Comparison of Traditional Spectroscopic and qPCR Methods

Moving forward in the next 3-4 years,we will likely see advanced development of thetraditional method into 24/7 commercially available real-time flow cytometry monitoring[13]. For qPCR,field prototypes using lab-on-a-chip technologies will appear feasible to be implemented for real-time monitoring[14-15].

Conclusions

The breakthrough application of fluorescence technologies to the identification of cyanobacteria is a promising tool to transform monitoring of cyanobacteria blooms in water systems to be rapid,and reliable over a 24/7 period.While the traditional spectroscopic method is well-known and utilized extensively in real-time 24/7 monitoring systems,the qPCR method using molecular techniques has advanced rapidly in the last decade.The qPCR′s ability to identify toxic cyanobacteria species individually or a few species (4-5) simultaneously,is certainly a key advantage.Both traditional spectroscopic and advanced qPCR technologies will continue to play centre stage in providing researchers,regulators and policy makers with important information on how to manage and prevent algal blooms in an increasingly uncertain climate change environment.

【編輯札記】對(duì)藻類(lèi)的在線(xiàn)預(yù)警一直是原水水質(zhì)管理人員關(guān)注的焦點(diǎn)，與傳統(tǒng)水廠(chǎng)工藝相比，預(yù)警技術(shù)仍處于發(fā)展期和摸索期，百家初放，各顯神通。將qPCR法和熒光光譜法作對(duì)比分析，客觀(guān)論述了兩種技術(shù)的術(shù)有專(zhuān)攻與應(yīng)用限制，本無(wú)優(yōu)劣之分，但對(duì)于不同需求的水庫(kù)管理，各有用武之地，能幫助我們更好地理解藻類(lèi)在線(xiàn)預(yù)警技術(shù)的進(jìn)展，為水庫(kù)管理者提供思路。