CN111792712A - A kind of method and application of inactivating enteritis virus in water - Google Patents

A kind of method and application of inactivating enteritis virus in water Download PDF

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CN111792712A
CN111792712A CN202010799475.7A CN202010799475A CN111792712A CN 111792712 A CN111792712 A CN 111792712A CN 202010799475 A CN202010799475 A CN 202010799475A CN 111792712 A CN111792712 A CN 111792712A
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water
virus
peracetic acid
inactivation
solution
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苑宝玲
唐爱茜
付明来
吴雪垠
艾慧颖
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Sibanze Ecological Environment Technology Xiamen Co ltd
Huaqiao University
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Huaqiao University
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    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00Treatment of water, waste water, or sewage
    • C02F1/50Treatment of water, waste water, or sewage by addition or application of a germicide or by oligodynamic treatment
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/002Grey water, e.g. from clothes washers, showers or dishwashers
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2303/00Specific treatment goals
    • C02F2303/04Disinfection

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Abstract

The invention provides a method for inactivating enterovirus in water and application thereof, belonging to the technical field of water purification treatment; the method comprises the following steps: adding 5-10 mg/L of peroxyacetic acid into water to be treated, and inactivating the enterovirus. The peracetic acid adopted by the invention can release nascent oxygen, and the nascent oxygen destroys virus genes through redox reaction and enables protein of the virus to be coagulated and denatured, thereby killing the virus. The method of the invention can overcome the problems of carcinogenesis, teratogenesis, mutagenesis and the like caused by inactivating viruses by using a chlorine-containing disinfectant, and can effectively kill the enterovirus in water.

Description

一种对水中肠道炎病毒灭活的方法和应用A kind of method and application of inactivating enteritis virus in water

技术领域technical field

本发明涉及水净化处理技术领域,尤其涉及一种对水中肠道炎病毒灭活的方法和应用。The invention relates to the technical field of water purification treatment, in particular to a method and application for inactivating enteritis virus in water.

背景技术Background technique

随着社会的发展,城市污水再生回用技术逐渐普及,同时再生水的安全问题逐渐引起人们的关注。城市污水通常含有的致病性强、易传染的病原微生物,若污水消毒过程没有将这些病原微生物完全灭活,那么在作为城市污水回用的再生水将会给公共健康带来巨大风险及威胁。而在城市污水中,肠道病毒广泛存在。目前,传统杀灭再生水中肠道炎病毒的方法多采用含氯消毒剂,含氯消毒剂具有致癌、致畸和致突变等缺陷。With the development of society, urban sewage recycling technology is gradually popularized, and the safety of recycled water has gradually attracted people's attention. Urban sewage usually contains highly pathogenic and contagious pathogenic microorganisms. If these pathogenic microorganisms are not completely inactivated during the sewage disinfection process, the reclaimed water reused as urban sewage will bring huge risks and threats to public health. In urban sewage, enteroviruses are widespread. At present, the traditional methods of killing enteritis viruses in regenerated water mostly use chlorine-containing disinfectants, which have defects such as carcinogenic, teratogenic and mutagenic.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种对水中肠道炎病毒灭活的方法和应用,本发明的方法能够克服含氯消毒剂灭活病毒导致的致癌、致畸和致突变等问题,而且能够有效杀灭水中肠道炎病毒。The object of the present invention is to provide a method and application for inactivating enteritis virus in water. The method of the present invention can overcome the problems of carcinogenesis, teratogenicity and mutagenesis caused by the inactivation of viruses by chlorine-containing disinfectants, and can effectively kill Determination of enteritis virus in water.

为了实现上述发明目的,本发明提供以下技术方案:In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

本发明提供了一种对水中肠道炎病毒灭活的方法,包括以下步骤:The invention provides a method for inactivating enteritis virus in water, comprising the following steps:

将待处理水pH调至4.5~8后,向待处理水中加入5~10mg/L的过氧乙酸,进行肠道炎病毒灭活。After the pH of the water to be treated is adjusted to 4.5-8, 5-10 mg/L of peracetic acid is added to the water to be treated to inactivate the enteritis virus.

优选的,所述水中肠道炎病毒包括轮状病毒和/或诺如病毒。Preferably, the water enteritis virus includes rotavirus and/or norovirus.

优选的,所述灭活的时间为1~35min。Preferably, the inactivation time is 1-35 min.

优选的,所述灭活的过程中伴随搅拌;所述搅拌的转速为200~300rpm。Preferably, the inactivation process is accompanied by stirring; the stirring speed is 200-300 rpm.

优选的,所述肠道炎病毒灭活后,还包括:调整灭活后水体的pH值至4.5~8,加入硫代硫酸钠-过氧化氢酶溶液淬灭灭活反应。Preferably, after the enteritis virus is inactivated, the method further includes: adjusting the pH value of the inactivated water body to 4.5-8, and adding a sodium thiosulfate-catalase solution to quench the inactivation reaction.

优选的,所述硫代硫酸钠-过氧化氢酶溶液以超纯水为溶剂,包括浓度为1.5~1.8mg/50mL的过氧化氢酶和浓度为25~35mg/50mL的硫代硫酸钠。Preferably, the sodium thiosulfate-catalase solution uses ultrapure water as a solvent, and includes catalase with a concentration of 1.5-1.8 mg/50 mL and sodium thiosulfate with a concentration of 25-35 mg/50 mL.

优选的,所述硫代硫酸钠-过氧化氢酶溶液的灭活后水体的体积比为1:1。Preferably, the volume ratio of the inactivated water body of the sodium thiosulfate-catalase solution is 1:1.

优选的,所述待处理的水包括城市污水。Preferably, the water to be treated includes municipal sewage.

本发明还提供了上述方案所述方法在污水处理或水质净化中的应用。The present invention also provides the application of the method described in the above scheme in sewage treatment or water purification.

本发明的有益效果:本发明提供了一种对水中肠道炎病毒灭活的方法,包括以下步骤:将待处理水pH调至4.5~8后,向待处理水中加入5~10mg/L的过氧乙酸,进行肠道炎病毒灭活。在本发明中,过氧乙酸能够释放出初生态氧,进而初生态氧通过氧化还原反应破坏病毒基因并使得病毒的蛋白质凝固变性。过氧乙酸自身分解为乙酸和氧气,无消毒副产物,保障水质安全,能够减少对人体健康的危害。而且过氧乙酸相较于传统氯消毒剂臭味较小,减弱了对人体的刺激和不适感;相较于臭氧消毒剂,制备简单易得;过氧乙酸在≤40℃的条件下每天分解率低于1%,相较于紫外线消毒方法,能够在较长的时间内发挥作用,有持续性杀菌能力。Beneficial effects of the present invention: The present invention provides a method for inactivating enteritis virus in water, comprising the following steps: after adjusting the pH of the water to be treated to 4.5-8, adding 5-10 mg/L of the water to be treated Peracetic acid for enteritis virus inactivation. In the present invention, peracetic acid can release nascent oxygen, and then nascent oxygen destroys viral genes through redox reactions and coagulates and denatures viral proteins. Peracetic acid is decomposed into acetic acid and oxygen by itself, without disinfection by-products, ensuring the safety of water quality and reducing the harm to human health. Moreover, compared with traditional chlorine disinfectants, peracetic acid has less odor, which reduces the irritation and discomfort to the human body; compared with ozone disinfectants, the preparation is simple and easy to obtain; peracetic acid decomposes every day under the condition of ≤40 °C The rate is lower than 1%. Compared with the ultraviolet disinfection method, it can play a role in a longer period of time and has continuous sterilization ability.

另外,本发明提出的一种过氧乙酸灭活肠道炎病毒的方法,补充了国内使用过氧乙酸对于肠道病毒消毒的系统研究,对城市回用中水消毒剂的剂量和水环境条件的调试具有重要意义。而且本发明的方法经济实用,无需增加或对现有工艺进行升级改造。无需大量的药剂,过氧乙酸的制备也易操作,没有传统臭氧、二氧化氯制备的工艺复杂。因此本发明提供的方法操作简单、经济实用。In addition, a method for inactivating enteritis virus by peracetic acid proposed by the present invention supplements the systematic research on enterovirus disinfection using peracetic acid in China, and the dosage and water environmental conditions of urban reused reclaimed water disinfectant are supplemented. debugging is important. Moreover, the method of the invention is economical and practical, and does not need to increase or upgrade the existing process. The preparation of peracetic acid is also easy to operate without requiring a large amount of pharmaceuticals, and it is not as complicated as the traditional preparation of ozone and chlorine dioxide. Therefore, the method provided by the present invention is simple to operate, economical and practical.

附图说明Description of drawings

图1为本发明实施例1中pH=7.8条件下过氧乙酸对猪轮状病毒灭活情况(PAA:5mg/L);Figure 1 shows the inactivation of porcine rotavirus by peracetic acid under the condition of pH=7.8 in Example 1 of the present invention (PAA: 5 mg/L);

图2为本发明实施例2中pH=7.8条件下过氧乙酸对杜兰病毒灭活情况(PAA:10mg/L);Figure 2 shows the inactivation of Duran virus by peracetic acid under the condition of pH=7.8 in Example 2 of the present invention (PAA: 10 mg/L);

图3为本发明实施例3中pH=5.4条件下过氧乙酸对猪轮状病毒灭活情况(PAA:10mg/L);Figure 3 shows the inactivation of porcine rotavirus by peracetic acid under the condition of pH=5.4 in Example 3 of the present invention (PAA: 10 mg/L);

图4为本发明实施例4中pH=4.5条件下过氧乙酸对杜兰病毒灭活情况(PAA:10mg/L)。Figure 4 shows the inactivation of Duran virus by peracetic acid under the condition of pH=4.5 in Example 4 of the present invention (PAA: 10 mg/L).

具体实施方式Detailed ways

本发明提供了一种对水中肠道炎病毒灭活的方法,包括以下步骤:将待处理水pH调至4.5~8后,向待处理水中加入5~10mg/L的过氧乙酸(PAA),进行肠道炎病毒灭活;所述待处理水pH优选的调整至7.8;用于调节所述待处理水pH的试剂优选为磷酸盐缓冲溶液。The invention provides a method for inactivating enteritis virus in water, comprising the following steps: after adjusting the pH of the water to be treated to 4.5-8, adding 5-10 mg/L peracetic acid (PAA) to the water to be treated , perform enteritis virus inactivation; the pH of the water to be treated is preferably adjusted to 7.8; the reagent for adjusting the pH of the water to be treated is preferably a phosphate buffer solution.

在本发明中,5~10mg/L的浓度在控制PAA的量的情况下又能达到好的消毒效果;所述待处理的水优选的包括城市污水;所述待处理水中的Co2+的浓度≤10-6mol/L;所述肠道炎病毒优选的包括轮状病毒和/或诺如病毒;所述轮状病毒优选的包括猪轮状病毒。In the present invention, the concentration of 5-10 mg/L can achieve a good disinfection effect under the condition of controlling the amount of PAA; the water to be treated preferably includes municipal sewage; the content of Co 2+ in the water to be treated is The concentration is less than or equal to 10 -6 mol/L; the enteritis virus preferably includes rotavirus and/or norovirus; the rotavirus preferably includes porcine rotavirus.

在本发明中,所述过氧乙酸的添加量优选为8mg/L;所述过氧乙酸优选的先溶解于的磷酸盐缓冲溶液中,得到过氧乙酸磷酸盐缓冲液,再将过氧乙酸磷酸盐缓冲液加入到水中;所述磷酸盐缓冲溶液的pH值优选为5、7、8;所述磷酸盐缓冲溶液的浓度优选为10mM;所述过氧乙酸磷酸盐缓冲液的浓度优选为20mg/L;在本发明具体实施过程中,过氧乙酸的浓度由Vacu-vials公司的K-7913过氧乙酸试剂盒检测,该试剂盒检测的浓度范围是0~5mg/L。In the present invention, the added amount of the peracetic acid is preferably 8 mg/L; the peracetic acid is preferably dissolved in the phosphate buffer solution first to obtain the peracetic acid phosphate buffer, and then the peracetic acid is dissolved in the phosphate buffer solution. Phosphate buffer is added to the water; the pH of the phosphate buffer is preferably 5, 7, 8; the concentration of the phosphate buffer is preferably 10mM; the concentration of the peracetic acid phosphate buffer is preferably 20 mg/L; in the specific implementation process of the present invention, the concentration of peracetic acid is detected by the K-7913 peracetic acid kit of Vacu-vials company, and the concentration range detected by the kit is 0-5 mg/L.

在本发明中,所述灭活的时间优选为1~35min,更优选为5~30min,最优选为10~20min;所述灭活的过程中优选的伴随搅拌;所述搅拌的转速优选为200~300rpm,更优选为250rpm。In the present invention, the inactivation time is preferably 1 to 35 minutes, more preferably 5 to 30 minutes, and most preferably 10 to 20 minutes; the inactivation process is preferably accompanied by stirring; the rotation speed of the stirring is preferably 200 to 300 rpm, more preferably 250 rpm.

在本发明中,过氧乙酸通过破坏轮状病毒和杜兰病毒的蛋白质外壳,导致过氧乙酸进入病毒内,进行遗传物质的破坏,从而使病毒失去感染力。In the present invention, peracetic acid destroys the protein shell of rotavirus and Duran virus, so that peracetic acid enters the virus and destroys the genetic material, thereby making the virus lose its infectivity.

本发明对肠道炎病毒灭活后,优选的还包括调整灭活后的溶液的pH至4.5~8,加入硫代硫酸钠-过氧化氢酶溶液淬灭灭活反应。在本发明中,所述调整后的pH值优选为6~7;所述硫代硫酸钠-过氧化氢酶溶液优选以超纯水为溶剂,包括浓度为1.5~1.8mg/50mL的过氧化氢酶和浓度为25~35mg/50mL的硫代硫酸钠;所述硫代硫酸钠的浓度优选为30mg/50mL。在本发明中,所述硫代硫酸钠-过氧化氢酶溶液的灭活后水体的体积比优选为1:1。在本发明中,硫代硫酸钠淬灭过氧乙酸生成醋酸,过氧化氢酶是用于淬灭过氧乙酸中含有的少量过氧化氢。After the enteritis virus is inactivated in the present invention, it preferably also includes adjusting the pH of the inactivated solution to 4.5-8, and adding a sodium thiosulfate-catalase solution to quench the inactivation reaction. In the present invention, the adjusted pH value is preferably 6 to 7; the sodium thiosulfate-catalase solution preferably uses ultrapure water as a solvent, including peroxide with a concentration of 1.5 to 1.8 mg/50 mL. Hydrogenase and sodium thiosulfate with a concentration of 25-35 mg/50 mL; the concentration of the sodium thiosulfate is preferably 30 mg/50 mL. In the present invention, the volume ratio of the inactivated water body of the sodium thiosulfate-catalase solution is preferably 1:1. In the present invention, sodium thiosulfate quenches peracetic acid to generate acetic acid, and catalase is used to quench a small amount of hydrogen peroxide contained in peracetic acid.

本发明在淬灭灭活反应后,还包括对淬灭灭活反应后的反应液进行稀释,稀释至水中病毒的浓度为101~102pfu/mL,得到稀释液,对稀释液进行病毒空斑计数实验;本发明对病毒空斑计数实验的具体过程没有特殊限制,以本领域常规病毒空斑计数实验流程为准即可;轮状病毒培养3天后计数,诺如病毒培养2天后计数。After the quenching and inactivation reaction, the present invention further includes diluting the reaction solution after the quenching and inactivation reaction, and diluting to the concentration of the virus in the water is 10 1 to 10 2 pfu/mL, to obtain the diluted solution, and performing the virus on the diluted solution. Plaque counting experiment; the present invention does not have special restrictions on the specific process of virus plaque counting experiment, and the routine virus plaque counting experiment procedure in the field can be used as the standard; rotavirus is counted after 3 days of culture, and norovirus is counted after 2 days of culture .

在本发明中,轮状病毒或诺如病毒需要及时进行计数。若取样后不能及时进行病毒空斑计数实验优选的放入-80℃冰箱内保存待测。In the present invention, rotavirus or norovirus needs to be counted in time. If the virus plaque counting experiment cannot be carried out in time after sampling, it is preferable to store it in a -80°C refrigerator for testing.

本发明还提供了上述方案所述方法在污水处理或水质净化中的应用。The present invention also provides the application of the method described in the above scheme in sewage treatment or water purification.

下面将结合本发明中的实施例,对本发明中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

实施例1Example 1

1)溶液配置1) Solution configuration

首先把含有105PFU/mL猪轮状病毒(ATCC-VR-892)的待处理水pH调至7.8,把调整好pH的水置于磁力搅拌器上,以200~300rpm的速度搅拌。First, the pH of the water to be treated containing 10 5 PFU/mL porcine rotavirus (ATCC-VR-892) was adjusted to 7.8, and the pH-adjusted water was placed on a magnetic stirrer and stirred at a speed of 200-300 rpm.

2)消毒过程2) Disinfection process

用pH为8的磷酸盐缓冲溶液配置浓度为20mg/L的过氧乙酸溶液。然后向步骤1)的水中投加配置好的过氧乙酸溶液,按照配置好的过氧乙酸溶液:待处理水=1:3的体积比加入,使得过氧乙酸的初始浓度达到5mg/L。反应5min和10min后分别取样,立刻分别加入0.5mL硫代硫酸钠-过氧化氢酶溶液(称取1.6mg过氧化氢酶(Catalase)和30mg硫代硫酸钠,溶于50mL超纯水中,灭活实验前,加入0.5mL的淬灭剂于1.7mL无菌离心管中)淬灭停止反应。对样品稀释后进行病毒空斑计数实验,3天后统计实验结果。A peracetic acid solution with a concentration of 20 mg/L was prepared with a phosphate buffer solution with a pH of 8. Then add the prepared peracetic acid solution to the water in step 1), and add it according to the volume ratio of prepared peracetic acid solution: water to be treated=1:3, so that the initial concentration of peracetic acid reaches 5 mg/L. After 5min and 10min of reaction, take samples respectively, immediately add 0.5mL sodium thiosulfate-catalase solution (we weigh 1.6mg catalase (Catalase) and 30mg sodium thiosulfate, dissolve in 50mL ultrapure water, Before the inactivation experiment, add 0.5 mL of quencher to a 1.7 mL sterile centrifuge tube) to quench to stop the reaction. After dilution of the sample, the virus plaque counting experiment was performed, and the experimental results were counted 3 days later.

病毒空斑计数实验具体流程如下:The specific process of virus plaque counting experiment is as follows:

(1)准备工作:(1) Preparations:

a)将细胞瓶中的病毒悬浮液接种到无菌6孔细胞培养板中,每个孔2mL,培养24h使细胞覆盖率达到95%形成单层细胞膜;a) Inoculate the virus suspension in the cell flask into a sterile 6-well cell culture plate, 2 mL per well, and culture for 24 hours to make the cell coverage reach 95% to form a monolayer cell membrane;

b)对待测病毒样品使用不含FBS的MEM培养液进行10倍梯度稀释至合适浓度;b) The virus sample to be tested is diluted to a suitable concentration by a 10-fold gradient with MEM culture medium without FBS;

c)按照表1配制半固体营养覆盖液。c) Prepare a semi-solid nutrient coating solution according to Table 1.

表1半固体营养覆盖液Table 1 Semi-solid nutrient coating solution

Figure BDA0002626865210000051
Figure BDA0002626865210000051

(2)空斑计数法实验步骤:(2) Experimental steps of plaque counting method:

a)使用不含FBS的MEM培养液清洗6孔细胞板中的单层细胞膜1次;a) Wash the monolayer cell membrane in the 6-well cell plate once with MEM medium without FBS;

b)缓慢加入450μL稀释后的病毒样品,轻轻晃动使其均匀分布在细胞膜上;b) Slowly add 450 μL of the diluted virus sample and shake it gently to distribute it evenly on the cell membrane;

c)将加入病毒样品后的6孔细胞板放入37℃、5%CO2的恒温培养箱中培养1h,期间每隔10min轻轻摇动细胞板使病毒充分均匀接触细胞膜;c) Put the 6-well cell plate after adding the virus sample into a constant temperature incubator at 37°C and 5% CO 2 for 1 hour, and gently shake the cell plate every 10 minutes to make the virus fully and evenly contact the cell membrane;

d)加入2.5mL上述半固体营养覆盖液,放入4℃冰箱中20min使其尽快冷却凝固;d) Add 2.5mL of the above-mentioned semi-solid nutrient covering solution, put it into a 4°C refrigerator for 20min to cool and solidify as soon as possible;

e)待覆盖液凝固后放入37℃、5%CO2的恒温培养箱中培养3d;e) After the covering liquid is solidified, put it into a constant temperature incubator at 37°C and 5% CO2 for 3 days;

f)培养后的细胞板中加入2mL 10%的甲醛固定液固定细胞,25℃放置3h;f) Add 2 mL of 10% formaldehyde fixative to the cultured cell plate to fix the cells, and place them at 25°C for 3 hours;

g)倒掉琼脂覆盖液,在孔内加4~5滴0.05%的结晶紫染液染色20min;g) Pour off the agar covering liquid, add 4 to 5 drops of 0.05% crystal violet staining solution to the well for 20 minutes;

h)使用去离子水将6孔细胞板中的多余的结晶紫洗净后进行计数:h) Use deionized water to wash the excess crystal violet in the 6-well cell plate and count:

病毒浓度=计数个数×稀释倍数。Virus concentration = number of counts × dilution factor.

结果参见图1。结果显示当Ct值达到18mg·min/L,过氧乙酸对猪轮状病毒的灭活对数为0.32,对应的灭活率为52.1%;当Ct值达到39mg·min/L,灭活对数为1.53,对应的灭活率为97.0%。See Figure 1 for the results. The results showed that when the Ct value reached 18 mg·min/L, the logarithm of inactivation of porcine rotavirus by peracetic acid was 0.32, and the corresponding inactivation rate was 52.1%. The number was 1.53, corresponding to an inactivation rate of 97.0%.

实施例2Example 2

1)溶液配置1) Solution configuration

首先把含有105PFU/mL杜兰病毒(ATCC-VR-1027,杜兰病毒与诺如病毒结构相似,故用杜兰病毒来展现诺如病毒的灭活表现)的待处理水pH调至7.8,把调整好pH的水置于磁力搅拌器上,以200~300rpm的速度搅拌。Firstly, the pH of the water to be treated containing 10 5 PFU/mL Duran virus (ATCC-VR-1027, Duran virus and Norovirus are similar in structure, so Duran virus is used to show the inactivation performance of Norovirus) is adjusted to 7.8. Put the pH-adjusted water on a magnetic stirrer and stir at a speed of 200-300 rpm.

2)消毒过程2) Disinfection process

用pH为8的磷酸盐缓冲溶液配置浓度为20mg/L的过氧乙酸溶液。然后向步骤1)的水中投加配置好的过氧乙酸溶液,按照配置好的过氧乙酸溶液:待处理水=1:1的体积比加入,使得过氧乙酸的初始浓度达到10mg/L。反应4、8、12和16min后取样,立刻分别加入0.5mL硫代硫酸钠-过氧化氢酶溶液淬灭停止反应。对样品稀释后进行病毒空斑计数实验,2天后统计实验结果。结果参见图2。结果显示当Ct值达到88mg·min/L,过氧乙酸对杜兰病毒的灭活对数为3.64,对应的灭活率为99.9%;当Ct值达到122mg·min/L,灭活对数为3.74,对应的灭活率为99.9%。A peracetic acid solution with a concentration of 20 mg/L was prepared with a phosphate buffer solution with a pH of 8. Then add the prepared peracetic acid solution to the water in step 1), and add it according to the volume ratio of prepared peracetic acid solution: water to be treated=1:1, so that the initial concentration of peracetic acid reaches 10 mg/L. Samples were taken after 4, 8, 12 and 16 min of reaction, and 0.5 mL of sodium thiosulfate-catalase solution was added immediately to quench and stop the reaction. After dilution of the sample, the virus plaque counting experiment was performed, and the experimental results were counted 2 days later. See Figure 2 for the results. The results showed that when the Ct value reached 88 mg·min/L, the inactivation logarithm of peracetic acid to Duran virus was 3.64, and the corresponding inactivation rate was 99.9%; when the Ct value reached 122 mg·min/L, the inactivation logarithm was 99.9%. was 3.74, corresponding to an inactivation rate of 99.9%.

实施例3Example 3

1)溶液配置1) Solution configuration

首先把含有105PFU/mL猪轮状病毒的待处理水pH调至5.4,把调整好pH的水置于磁力搅拌器上,以200~300rpm的速度搅拌。Firstly, the pH of the water to be treated containing 10 5 PFU/mL porcine rotavirus was adjusted to 5.4, and the pH-adjusted water was placed on a magnetic stirrer and stirred at a speed of 200-300 rpm.

2)消毒过程2) Disinfection process

用pH为5的磷酸盐缓冲溶液配置浓度为20mg/L的过氧乙酸溶液。然后向步骤1)的水中投加配置好的过氧乙酸溶液,按照配置好的过氧乙酸溶液:待处理水=1:1的体积比加入,使得过氧乙酸的初始浓度达到10mg/L。反应1、2、4和8min后取样,立刻分别加入0.5mL硫代硫酸钠-过氧化氢酶溶液淬灭停止反应。对样品稀释后进行病毒空斑计数实验,3天后统计实验结果。结果参见图3。结果显示当Ct值达到31mg·min/L,过氧乙酸对猪轮状病毒的灭活对数为2.96,对应的灭活率为99.89%;当Ct值达到63mg·min/L,灭活对数为5.51,对应的灭活率为99.999%。A peracetic acid solution with a concentration of 20 mg/L was prepared with a phosphate buffer solution with a pH of 5. Then add the prepared peracetic acid solution to the water in step 1), and add it according to the volume ratio of prepared peracetic acid solution: water to be treated=1:1, so that the initial concentration of peracetic acid reaches 10 mg/L. Samples were taken after 1, 2, 4 and 8 minutes of reaction, and 0.5 mL of sodium thiosulfate-catalase solution was added immediately to quench and stop the reaction. After dilution of the sample, the virus plaque counting experiment was performed, and the experimental results were counted 3 days later. See Figure 3 for the results. The results showed that when the Ct value reached 31 mg·min/L, the logarithm of inactivation of porcine rotavirus by peracetic acid was 2.96, and the corresponding inactivation rate was 99.89%. The number was 5.51, corresponding to an inactivation rate of 99.999%.

实施例4Example 4

1)溶液配置1) Solution configuration

首先把含有105PFU/mL杜兰病毒的待处理水pH调至4.5,把调整好pH的水置于磁力搅拌器上,以200~300rpm的速度搅拌。Firstly, the pH of the water to be treated containing 10 5 PFU/mL of Duran virus was adjusted to 4.5, and the pH-adjusted water was placed on a magnetic stirrer and stirred at a speed of 200-300 rpm.

2)消毒过程2) Disinfection process

用pH为5的磷酸盐缓冲溶液配置浓度为20mg/L的过氧乙酸溶液。然后向步骤1)的水中投加配置好的过氧乙酸溶液,按照配置好的过氧乙酸溶液:待处理水=1:1的体积比加入,使得过氧乙酸的初始浓度达到10mg/L。反应4、8、16、24和35min后取样,立刻分别加入0.5mL硫代硫酸钠-过氧化氢酶溶液淬灭停止反应。对样品稀释后进行病毒空斑计数实验,2天后统计实验结果。结果参见图4。结果显示当Ct值达到234mg·min/L,过氧乙酸对杜兰病毒的灭活对数为3.00,对应的灭活率为99.9%;当Ct值达到378mg·min/L,灭活对数为3.70,对应的灭活率为99.98%。A peracetic acid solution with a concentration of 20 mg/L was prepared with a phosphate buffer solution with a pH of 5. Then add the prepared peracetic acid solution to the water in step 1), and add it according to the volume ratio of prepared peracetic acid solution: water to be treated=1:1, so that the initial concentration of peracetic acid reaches 10 mg/L. Samples were taken after 4, 8, 16, 24 and 35 minutes of reaction, and 0.5 mL of sodium thiosulfate-catalase solution was added immediately to quench and stop the reaction. After dilution of the sample, the virus plaque counting experiment was performed, and the experimental results were counted 2 days later. See Figure 4 for the results. The results showed that when the Ct value reached 234 mg·min/L, the inactivation logarithm of peracetic acid to Duran virus was 3.00, and the corresponding inactivation rate was 99.9%; when the Ct value reached 378 mg·min/L, the inactivation logarithm was 378 mg·min/L. is 3.70, corresponding to an inactivation rate of 99.98%.

实施例5Example 5

1)溶液配置1) Solution configuration

首先把含有105PFU/mL猪轮状病毒的待处理水pH调至7.8,把调整好pH的水置于磁力搅拌器上,以200~300rpm的速度搅拌。Firstly, the pH of the water to be treated containing 10 5 PFU/mL porcine rotavirus was adjusted to 7.8, and the pH-adjusted water was placed on a magnetic stirrer and stirred at a speed of 200-300 rpm.

2)消毒过程2) Disinfection process

用pH为8的磷酸盐缓冲溶液配置浓度为20mg/L的过氧乙酸溶液。然后向步骤(1)的水中投加配置好的过氧乙酸溶液,按照配置好的过氧乙酸溶液:待处理水=1:1的体积比加入,使得过氧乙酸的初始浓度达到10mg/L。反应16min后取样,立刻分别加入0.5mL硫代硫酸钠-过氧化氢酶溶液淬灭停止反应。进行两步法RT-qPCR检测。A peracetic acid solution with a concentration of 20 mg/L was prepared with a phosphate buffer solution with a pH of 8. Then add the configured peracetic acid solution to the water in step (1), and add it according to the configured peracetic acid solution: water to be treated=1:1 volume ratio, so that the initial concentration of peracetic acid reaches 10 mg/L . After 16 minutes of reaction, samples were taken, and 0.5 mL of sodium thiosulfate-catalase solution was added immediately to quench and stop the reaction. A two-step RT-qPCR assay was performed.

(1)实验试剂(1) Experimental reagents

OSU逆转录(RT)引物:VP7-R,qPCR引物VP7-R、VP7-FOSU reverse transcription (RT) primers: VP7-R, qPCR primers VP7-R, VP7-F

TV逆转录(RT)引物:VP2-R,qPCR引物NSP-R1、NSP1-F1TV reverse transcription (RT) primers: VP2-R, qPCR primers NSP-R1, NSP1-F1

表4.1引物一览表Table 4.1 List of primers

Figure BDA0002626865210000081
Figure BDA0002626865210000081

(2)实验步骤(2) Experimental steps

通过逆转录酶根据碱基互补配对法则将核糖核酸(Ribonucleic Acid,RNA)逆转录为脱氧核糖核酸(DeoxyribonucleicAcid,DNA),即互补DNA(cDNA)后,使用聚合酶链式反应来扩增出大量与cDNA对应的DNA。最后使用定量聚合酶链式反应(Quantitativepolymerase chain reaction,qPCR)通过荧光染料表征出DNA浓度并得到线性关系,再来定量测量扩增后的DNA的荧光强度表征浓度。Ribonucleic Acid (RNA) is reverse transcribed into Deoxyribonucleic Acid (DNA) by reverse transcriptase according to the base complementary pairing rule, that is, after complementary DNA (cDNA), polymerase chain reaction is used to amplify a large number of DNA corresponding to cDNA. Finally, quantitative polymerase chain reaction (quantitative polymerase chain reaction, qPCR) was used to characterize the DNA concentration through fluorescent dyes and a linear relationship was obtained, and then the fluorescence intensity of amplified DNA was quantitatively measured to characterize the concentration.

结果显示:过氧乙酸对于猪轮状病毒和杜兰病毒的灭活机理相似,即破坏两种病毒的蛋白质外壳,导致过氧乙酸进入病毒内进行遗传物质的破坏,从而使病毒失去感染力。The results showed that the inactivation mechanism of peracetic acid for porcine rotavirus and Tulane virus is similar, that is, destroying the protein shell of the two viruses, causing peracetic acid to enter the virus to destroy the genetic material, thus making the virus lose its infectivity.

以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

序列表sequence listing

<110> 华侨大学<110> Huaqiao University

<120> 一种对水中肠道炎病毒灭活的方法和应用<120> A method and application for inactivating enteritis virus in water

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Claims (9)

1.一种对水中肠道炎病毒灭活的方法,包括以下步骤:1. A method for inactivating enteritis virus in water, comprising the following steps: 将待处理水pH调至4.5~8后,向待处理水中加入5~10mg/L的过氧乙酸,进行肠道炎病毒灭活。After the pH of the water to be treated is adjusted to 4.5-8, 5-10 mg/L of peracetic acid is added to the water to be treated to inactivate the enteritis virus. 2.根据权利要求1所述的方法,其特征在于,所述水中肠道炎病毒包括轮状病毒和/或诺如病毒。2. The method according to claim 1, wherein the water enteritis virus comprises rotavirus and/or norovirus. 3.根据权利要求1所述的方法,其特征在于,所述灭活的时间为1~35min。3 . The method according to claim 1 , wherein the inactivation time is 1-35 min. 4 . 4.根据权利要求1所述的方法,其特征在于,所述灭活的过程中伴随搅拌;所述搅拌的转速为200~300rpm。4 . The method according to claim 1 , wherein the inactivation is accompanied by stirring; the stirring speed is 200-300 rpm. 5 . 5.根据权利要求1所述的方法,其特征在于,所述肠道炎病毒灭活后,还包括:调整灭活后水体的pH值至4.5~8,加入硫代硫酸钠-过氧化氢酶溶液淬灭灭活反应。5. The method according to claim 1, characterized in that, after the enteritis virus is inactivated, further comprising: adjusting the pH value of the water body after the inactivation to 4.5-8, adding sodium thiosulfate-hydrogen peroxide The enzyme solution quenches the inactivation reaction. 6.根据权利要求5所述的方法,其特征在于,所述硫代硫酸钠-过氧化氢酶溶液以超纯水为溶剂,包括浓度为1.5~1.8mg/50mL的过氧化氢酶和浓度为25~35mg/50mL的硫代硫酸钠。6. The method according to claim 5, wherein the sodium thiosulfate-catalase solution uses ultrapure water as a solvent, comprising catalase with a concentration of 1.5-1.8 mg/50mL and a concentration of It is 25~35mg/50mL of sodium thiosulfate. 7.根据权利要求5所述的方法,其特征在于,所述硫代硫酸钠-过氧化氢酶溶液的灭活后水体的体积比为1:1。7 . The method according to claim 5 , wherein the volume ratio of the water body after the deactivation of the sodium thiosulfate-catalase solution is 1:1. 8 . 8.根据权利要求1所述的方法,其特征在于,所述待处理的水包括城市污水。8. The method of claim 1, wherein the water to be treated comprises municipal sewage. 9.权利要求1~8任意一项所述方法在污水处理或水质净化中的应用。9. Application of the method of any one of claims 1 to 8 in sewage treatment or water purification.
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