CN111485014A

CN111485014A - Method for detecting live bacteria of escherichia coli

Info

Publication number: CN111485014A
Application number: CN201910075780.9A
Authority: CN
Inventors: 王加启; 郑楠; 董蕾; 刘慧敏; 孟璐
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2020-08-04

Abstract

In order to solve the problems of false positive and false negative when the PMA-qPCR method is adopted to detect the viable bacteria in the prior art, the invention provides an improved method for detecting and determining the viable bacteria of escherichia coli.

Description

Method for detecting live bacteria of escherichia coli

Technical Field

The invention belongs to the field of microbial detection, and relates to a method for detecting live bacteria of escherichia coli.

Background

Escherichia coli is a high-risk pathogenic bacterium, is very closely related to our daily life, and is usually parasitic in livestock manure or in water contaminated with manure. When people eat food polluted by escherichia coli, symptoms such as parenteral infection, acute diarrhea and the like can be caused, and symptoms such as fever, vomiting and the like are accompanied. Most of the worldwide cases of E.coli infectious diseases are caused by food transmission.

Conventional methods for detecting Escherichia coli, such as multitubular fermentation, filtration, plate counting, and the like, are inexpensive and reliable, but are laborious and time-consuming. PCR-based methods can overcome these disadvantages of conventional methods due to their specificity and sensitivity. In particular, real-time fluorescent quantitative pcr (qpcr) can detect e.coli in milk samples in real-time or rapidly. However, using qPCR it is difficult to distinguish between live and dead cells, which results in an overestimation of the number of live cells, especially when the number of dead cells exceeds the number of live cells.

Therefore, when the qPCR method is adopted to detect the viable bacteria of the escherichia coli in the milk, the problems of false negative and false positive exist. PMA (azido propyl bromide) -qPCR detection technology and EMA (azido ethidium bromide) -qPCR are viable bacteria detection technology which is started in recent years, and the principle is that a photoreactive dye PMA or EMA and the like which is impermeable to cell membranes and has high DNA affinity are designed to penetrate the cell membranes of damaged cells, the PMA or EMA is embedded into double-stranded DNA, the double-stranded DNA is exposed to strong visible light to form covalent bond modified DNA, and the amplification of the DNA in the PCR reaction is inhibited, so that the purposes of distinguishing dead bacteria and viable bacteria are achieved.

However, the PMA-qPCR or EMA-qPCR detection result is found to be higher than the actual viable bacteria value, which means that part of the DNA of the non-viable bacteria is amplified in the qPCR reaction. PMA-qPCR or EMA-qPCR detection technology can not effectively distinguish live bacteria from dead bacteria when detecting live bacteria treated at low temperature, and the conditions of high detection value and inaccurate detection result are easy to occur.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for detecting live bacteria of escherichia coli, which adopts a real-time fluorescence quantitative PCR (qPCR) method, can accurately detect the number of live cells of the escherichia coli in a sample and has high sensitivity.

The invention provides the following technical scheme:

a method for detecting the number of viable bacteria in Escherichia coli comprises the following steps: before extracting the DNA of the thallus, the thallus is treated by using a photoreaction dye with DNA affinity of cell membrane impermeability, the unreacted photoreaction dye is removed after the light irradiation reaction, the DNA is extracted to carry out real-time fluorescence quantitative PCR (qPCR), and when the qPCR is carried out, the DNA of the bacillus cereus is added as an amplification internal reference.

According to the invention, the upstream primer sequence of the amplification internal reference is as follows: 5'-CGCAAGGCTGAAACTCAAAG-3' (SEQ ID NO.1), the sequence of the downstream primer is: 5'-GAGGATGTCAAGACCTGGTAAG-3' (SEQ ID NO. 2).

According to the invention, the hydrolysis probe sequence for the bacillus cereus DNA fragment is: 5'-ACAAGCGGTGGAGCATGTGGTTTA-3' (SEQ ID NO. 3).

According to the invention, when qPCR is carried out, the sequence of the upstream primer for amplifying the escherichia coli is as follows: 5'-GGTAGAGCACTGTTTTGGCA-3' (SEQ ID NO.4), the sequence of the downstream primer is: 5'-TGTCTCCCGTGATAACTTTCTC-3' (SEQ ID NO. 5).

According to the invention, the hydrolysis probe sequence for the E.coli DNA is: 5'-TCATCCCGACTTACCAACCCG-3' (SEQ ID NO. 6).

According to the present invention, the method comprises the step of adding SDS to the cells before treating the cells with the photoreactive dye.

As an embodiment of the invention, the SDS is added to a final concentration of 50-150ppm, for example 90-110 ppm. In one embodiment of the invention the final concentration of SDS after addition is 100 ppm.

According to the invention, the photoreactive dye may be PMA or EMA. In one embodiment of the invention the photoreactive dye is PMA.

Preferably, the photoreactive dye is added to a final concentration of 30 to 50. mu.M, more preferably 35 to 45. mu.M. In one embodiment of the invention the final concentration of the photoreactive dye after addition is 40. mu.M.

In one embodiment of the present invention, the light reaction is processed in the following manner: the cells and the photoreactive dye are mixed well, incubated in the dark for 15 to 25 minutes, for example, 20 minutes, and then exposed to a 500W tungsten lamp for 5 to 15 minutes, for example, 10 minutes.

In one embodiment of the present invention, the reaction conditions of the qPCR are:

the qPCR reaction (20. mu. L) contained 10. mu. L KAPA PROBE FAST qPCR MasterMix, 1. mu. L DNA template, 0.05. mu. L (250nM) for each of the upstream and downstream primers, and 0.1. mu. L (500nM) for the hydrolysis PROBE.

Reaction procedure: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 1min, and 40 cycles.

According to the present invention, the step of extracting the DNA of the bacterial cells is to extract the DNA by the cetyltrimethylammonium bromide (CTAB) method.

According to the invention, the method can be used for detecting the number of live bacteria of escherichia coli in milk or drinking water.

According to the invention, the method also comprises a dairy pretreatment step. The pretreatment step is to enrich Escherichia coli in milk. In one embodiment of the present invention, the dairy product pretreatment step comprises centrifuging the dairy product, removing upper fat and supernatant, washing the pellet, and resuspending the pellet. In one embodiment of the present invention, the conditions of the centrifugation are: centrifuging at 10000r/min for 5-10 min. In one embodiment of the invention, the pellet is washed with sterile saline and resuspended.

According to the invention, the method also comprises the step of preparing a standard curve of the bacterial number and the qPCR Cq value by using the Escherichia coli liquid with known concentration and gradient dilution. In one embodiment of the present invention, the step of preparing the standard curve comprises: taking the viable bacteria suspension of the escherichia coli, and properly diluting the viable bacteria suspension to ensure that the concentration of the bacteria liquid is 10⁷CFU/m L, then extracting DNA, diluting the extracted DNA for 5 times according to a 10-fold gradient, performing qPCR reaction on the DNA at each concentration, and calculating by using CFXManager 3.1 to obtain a standard curve.

According to the invention, the method for removing the unreacted photoreaction dye comprises the steps of centrifuging the thallus mixture after the photoreaction and washing the precipitate. In one embodiment of the invention, the centrifugation conditions are 10000g for 20min and the washing is performed with PBS.

The primer of the Escherichia coli is proved to have good inclusion and exclusivity by 14 strains.

Advantageous effects

(1) In the invention, during qPCR, the DNA of the bacillus cereus is used as an internal reference gene, and the DNA has complete heterogeneity with the escherichia coli to be detected, thereby avoiding the occurrence of false negative results. Because the DNA of the bacillus cereus is very stable and is not easy to decompose, the effective action of adding internal parameters is ensured.

(2) According to the invention, before the bacteria are treated by the photoreaction dye, SDS is used to enhance the permeability of the photoreaction dye to the bacteria of Escherichia coli. SDS is very effective in membrane destabilization and can be used to accurately distinguish between live and dead cells. The inventors found through experiments that SDS has a better effect of promoting the permeability of photoreactive dyes than other surfactants such as lecithin and the like. However, SDS also damages cell membranes of living cells, and the concentration of SDS needs to be selected to ensure that the permeation promoting effect of SDS and the damage effect on living cells are properly balanced.

(3) The invention can accurately detect the number of the live Escherichia coli cells in the milk by adopting the photoreaction dye with lower concentration than the prior art, greatly reduces the cost and can still keep good sensitivity.

(4) The invention uses Cetyl Trimethyl Ammonium Bromide (CTAB) method to extract DNA, the extraction efficiency of the method is higher than that of the kit method, the concentration of the extracted DNA is higher, and the sensitivity of the detection method can be further improved.

(5) The method can quickly detect the live Escherichia coli in the milk, has strong specificity and high sensitivity, can avoid false positive results, and further can avoid false negative results.

Definition and description of terms

The term "qPCR" generally refers to what is known as real-time quantitative polymerase chain reaction. This technique uses PCR to simultaneously amplify and quantify target nucleic acids, where quantification relies on the insertion of a fluorescent dye or sequence specific probe containing a fluorescent reporter molecule that is detectable only after hybridization to the target nucleic acid.

The simplest method of quantification is to use an intercalated fluorescent dye, such as SYBR green or EVA green. These dyes insert themselves into the double stranded DNA molecules generated during extension of a particular product. As the amount of PCR product increases, more dye is incorporated and the fluorescence signal increases.

Another way of quantifying is to use sequence specific probes, either hydrolysis (TaqMan) or hybridization (L light-Cycler) probes, the hydrolysis probe is labeled with a fluorescent dye at the 5 'end and a quencher at the 3' end, quenching of the fluorescent signal is caused by the spatial proximity of the quencher to the reporter dye, the hydrolysis probe is cleaved off during the synthesis of complementary DNA during the extension phase, the reporter dye and quencher are separated and fluoresce.

Frequently used fluorescent dyes include, but are not limited to, Fluorphor 1, Fluorphor2, aminocoumarin, fluorofluorescein, Cy3, Cy5, europium, terbium, BODIPY, dansyl, naphthalene (naphalene), ruthenium, tetramethylrhodamine, 6-carboxyfluorescein (6-FAM), VIC, YAK, rhodamine, and texas red (TexasRed). Quenchers often used include, but are not limited to: TAMRATM, 6-carboxytetramethylrhodamine, methyl Red or dark quencher (dark quencher).

The term "real-time" refers to a specific measurement being taken at each cycle of PCR. An increase in target sequence correlates with an increase or decrease in fluorescence from cycle to cycle. At the end of a run (usually consisting of several cycles), quantification is performed based on the resulting fluorescent signal during the exponential phase of the PCR. Measurement of amplification is typically done by a Cq (quantitative cycle) value, which describes the first rise in fluorescence to a cycle value significantly above background fluorescence.

The term "membrane impermeable DNA affinity photoreactive dye" has the technical meaning common in the art. They are cell membrane impermeable, but have a high affinity for DNA, and can penetrate into cells with damaged cell membranes, intercalate into their double stranded DNA, and covalently bind to DNA under visible light. Known such photoreactive dyes include, but are not limited to, PMA (azido-propylidene bromide), EMA (azido-ethidium bromide).

The term "milk" includes, but is not limited to, dairy products or dairy products such as raw milk, liquid milk, sterilized milk, and the like, e.g., milk derived from human or mammalian secretion, including, but not limited to, cow's milk, goat's milk, horse's milk, donkey's milk, camel's milk, human milk, and the like, as well as products prepared using such milk.

Drawings

FIG. 1 shows the inhibition of viable bacteria of Escherichia coli by SDS concentration, and the abscissa of the graph is 3 × 10⁷The SDS concentration added to the CFU/m L E.coli solution was plotted on the ordinate against the number of colonies after plating.

FIG. 2 is the effect of PMA concentration on the Cq value of qPCR. The abscissa is the concentration of PMA added before DNA extraction and the ordinate is the Cq value of qPCR.

FIG. 3 is a schematic representation at 1 × 10²-1×10⁷And (3) extracting a standard curve of the corresponding DNA concentration and the Cq value of qPCR under the CFU/m L bacterial concentration.

FIG. 4 shows the results of detecting the number of viable bacteria of Escherichia coli in a sample subjected to the SDS-PMA-qPCR method. The group a is a milk sample added with live bacteria, the group b is a milk sample added with live bacteria and dead bacteria, and the group c is a milk sample added with live bacteria and dead bacteria. The black column was SDS and PMA added, and the gray column was SDS and PMA not added.

Detailed Description

The present invention is further described below with reference to examples. It should be noted that the examples are not intended to limit the scope of the present invention, and those skilled in the art will appreciate that any modifications and variations based on the present invention are within the scope of the present invention.

The chemical reagents used in the following examples are conventional and are commercially available.

The general experimental methods used in the following examples are as follows:

1. cultivation of the Strain

Escherichia coli (ATCC25922) and Bacillus cereus (ATCC11778) were inoculated into L B culture medium, respectively, and incubated at 37 ℃ for 24 hours in a shaker, 10 ml of the suspension was transferred to a 50 ml kaning tube, centrifuged at 15000g at 4 ℃ for 3 minutes, the supernatant was decanted, and resuspended in 0.85% physiological saline, and in order to determine the concentration of the bacterial solution, it was diluted in 6 steps in sequence with physiological saline, 100. mu. L was applied to L B agar, and the resultant was incubated at 37 ℃ for 24 hours and counted.

Other strains for experiments were cultured in the same manner.

2. DNA extraction

The cells were resuspended in TE buffer (10mM Tris-HCl and 1mM EDTA, pH 8.0), a stock solution of lysostaphin (1000. mu.g/m L, deionized water as solvent) was added to the cells and the final concentration was adjusted to 200. mu.g/m L, the mixture was incubated at 37 ℃ for 1 hour, DNA extraction was carried out by the cetyltrimethylammonium bromide (CTAB) method, and the extracted DNA was stored at-20 ℃ after concentration measurement with a Nanodrop 1000 spectrophotometer.

3. Preparation of dead bacteria

The bacterial suspension was centrifuged at 5000g at 4 ℃ for 10min, the supernatant was decanted off, and the bacteria were resuspended in 0.1% peptone water. The concentration of the resuspended liquid was adjusted to 10⁷-10⁸CFU/m L, and dividing into two parts, wherein one part is heated at 90 deg.C for 20min to prepare dead bacteria, and the other part is used as live bacteria.

Example 1 verification of primer inclusion and exclusivity

Bacterial DNA was extracted by the CTAB method using 14 strains, including 7 E.coli standard strains and another 7 known strains, PCR reaction system (25. mu. L) including 12.5. mu. L Master Mix, template DNA 2. mu. L, upstream and downstream primers each 1. mu. L, primer concentration 4000nM, water 8.5. mu. L.

The primer sequence is as follows: an upstream primer 5'-GGTAGAGCACTGTTTTGGCA-3', a downstream primer 5'-TGTCTCCCGTGATAACTTTCTC-3';

the reaction procedure was pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 1min, and 40 cycles, the amplification products were electrophoresed in 2.5% agarose gel electrophoresis in 1 × TE buffer.

As shown in Table 1, only the E.coli DNA showed a positive signal and the other species did not show a positive signal with the primers. This result indicates that the selected primers are highly specific and do not interfere with the DNA of other species.

TABLE 1 strains for the test of inclusion and exclusion

Example 2 optimization of SDS concentration

SDS was dissolved in 0.1% peptone water to prepare 20% SDS stock solution, which was then sterilized.

Escherichia coli (ATCC25922) bacterial liquid (10)⁷CFU/m L) at 4 ℃ for 10min at 5000g, then resuspending with SDS of different concentrations, the concentration of SDS is 0, 25, 50, 100, 250, 500, 1000ppm respectively, 100 mu L solution is taken and coated on a plate, cultured for 24h at 37 ℃, the colony number is observed, and the optimal concentration is selected according to the inhibition condition of SDS on the live bacteria of escherichia coli.

As shown in fig. 1, when the SDS concentration was 0, 25, 50, 100ppm, the corresponding logCFU values were 7.07, 7.06, respectively. The logCFU values were 6.95, 6.93, 6.89, 6.79 at SDS concentrations of 200, 250, 500, 1000ppm, respectively. As can be seen from the figure, there is a sharp decrease in logCFU value at SDS concentrations of 100-250 ppm. Therefore, 100ppm was used as the optimum concentration for inhibiting the signal of dead cells.

EXAMPLE 3 optimization of PMA concentration

PMA was dissolved in sterile water to prepare a 10mM stock solution, which was stored at-20 ℃ in the dark.

Before PMA treatment, an equal volume of viable E.coli (ATCC25922) liquid (10) was added⁷CFU/m L) and sterilized solution (10)⁷CFU/M L), adding 2M L mixed solution into a centrifuge tube, adding selected SDS solution with optimal concentration, adding PMA solution with different concentrations of 0, 10, 20, 30, 40 and 50 mu M respectively, mixing well, and then addingCulturing in dark for 20min to make PMA enter damaged cells to the maximum extent. The tube was then exposed to light at 20cm under a 500W tungsten lamp for 10min (tube opening up, on ice). The exposed liquid was centrifuged at 10000g for 20min to remove PMA and washed with PBS. Subsequently, genomic DNA was extracted by CTAB method and qPCR detection was performed.

The qPCR reaction system (20. mu. L) comprises 10. mu. L KAPA PROBE FAST qPCR Master Mix, E.coli DNA template and IAC DNA template each 1. mu. L, upstream and downstream primers each 250nM, volume of 0.05. mu. L, PROBEs each 500nM, volume of 0.1. mu. L, and the maximum Cq value corresponding to PMA concentration is selected as the optimum concentration.

The primer sequence is as follows: an upstream primer 5'-GGTAGAGCACTGTTTTGGCA-3', a downstream primer 5'-TGTCTCCCGTGATAACTTTCTC-3'; the hydrolysis probe sequence is: 5'-TCATCCCGACTTACCAACCCG-3' are provided.

The IAC primer sequence is as follows: an upstream primer 5'-CGCAAGGCTGAAACTCAAAG-3', a downstream primer 5'-GAGGATGTCAAGACCTGGTAAG-3'; the hydrolysis probe sequence is: 5'-ACAAGCGGTGGAGCATGTGGTTTA-3' are provided.

Reaction procedure: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 1min, and performing 40 cycles, wherein fluorescence signals are collected.

As shown in fig. 2, when the PMA concentration is 0, 10, 20, 30, 40, 50 μ M, the corresponding Cq values are 20.45, 20.68, 20.73, 21.45, 23.68, 23.12, respectively, and the optimum concentration of PMA is 40 μ M.

Example 4

qPCR reaction (20. mu. L) 10. mu. L KAPA PROBE FAST qPCR Master Mix, 1. mu. L DNA template, 250nM each of the up and down primers, 0.05. mu. L each, 500nM each of the PROBEs, 0.1. mu. L each, primers and reaction program identical to the PCR in example 1.

The DNA of Bacillus cereus was used as IAC, and the primer and probe sequences were the same as those in example 1.

The addition of the internal reference in the reaction system can avoid false negative results, for example, if the result of Escherichia coli is negative after amplification reaction, and the result of Bacillus cereus is positive, the result of Escherichia coli is abnormal, and the problems of PCR reaction instruments and systems can be eliminated.

The qPCR amplification reaction was performed according to the qPCR procedure described in this example, and a standard curve was calculated using CFX Manager 3.1.

Example 5 detection of viable Escherichia coli in a spiked sample by SDS-PMA-qPCR

The experimental use of Mongolian cattle UHT milk purchased from supermarket, the sterility is verified by flat plate method, the milk is divided into three groups, namely three groups a, b and c, each group is divided into two parts, the group a is added with Escherichia coli ATCC25922 live bacteria liquid, the final volume is 2m L, and the final concentration of live bacteria is 3 × 10²CFU/m L, adding viable bacteria solution and dead bacteria solution of Escherichia coli ATCC25922 to make final volume 2m L and final concentration of viable bacteria 3 × 10²CFU/m L, and the final concentration of dead bacteria is 3 × 10³CFU/m L, adding viable bacteria solution and dead bacteria solution of Escherichia coli ATCC25922 with final volume of 2m L and viable bacteria concentration of 3 × 10³CFU/m L, and the final concentration of dead bacteria is 3 × 10²CFU/m L. all samples were in triplicate. before DNA extraction, treatments with and without SDS and PMA were set up, respectively.

As shown in FIG. 4, SDS-PMA-qPCR was used to detect viable E.coli in a spiked sample to evaluate the efficiency of the method, as shown in a of FIG. 4, 3 × 10 was added to a milk sample²CFU/m L viable bacteria, the Cq value with SDS and PMA added was 32.15, and the Cq value without SDS and PMA was 31.78, as shown in b of FIG. 4, when 3 × 10 was mixed in the milk sample²Live bacteria of CFU/m L and 3 × 10³When CFU/m L was killed, the Cq value was 32.35 when SDS and PMA were added and 29.82 when PMA and SDS were not added, the same trend was observed when 3 × 10 was added to the milk sample as shown in c in FIG. 4³CFU/m L live bacteria and 3 × 10²When CFU/m L was killed, the Cq value was 29.78 when PMA and SDS were added, and 28.67 when PMA and SDS were not added.

The above examples fully demonstrate that the method has high specificity and high sensitivity, and can accurately detect live escherichia coli in milk. By adding SDS and PMA with proper concentration before DNA extraction, the false positive signal can be completely eliminated, and by using IAC, the false negative result can be eliminated, so that said method can simultaneously prevent false positive result and false negative result from appearing.

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Claims

1. A method for detecting the number of viable bacteria in Escherichia coli comprises the following steps: before extracting thallus DNA, treating thallus with cell membrane impermeable light reaction dye with DNA affinity, after light reaction removing unreacted light reaction dye, extracting DNA and real-time fluorescent quantitative PCR, its characteristic is: during the qPCR, the DNA of Bacillus cereus was added as an internal reference for amplification.

2. The method of claim 1, wherein the upstream primer sequence of the bacillus cereus DNA is: 5'-CGCAAGGCTGAAACTCAAAG-3', the sequence of the downstream primer is as follows: 5'-GAGGATGTCAAGACCTGGTAAG-3', respectively;

preferred hydrolysis probe sequences for use with bacillus cereus are: 5'-ACAAGCGGTGGAGCATGTGGTTTA-3' are provided.

3. The method of any one of claims 1 or2, wherein the qPCR is performed with an upstream primer sequence that amplifies e.coli: 5'-GGTAGAGCACTGTTTTGGCA-3', the sequence of the downstream primer is as follows: 5'-TGTCTCCCGTGATAACTTTCTC-3', respectively;

preferred hydrolysis probe sequences for use in E.coli are: 5'-TCATCCCGACTTACCAACCCG-3' are provided.

4. A method according to any of claims 1 to 3, wherein the photoreactive dye is added to a final concentration of 30 to 50 μ M, more preferably 35 to 45 μ M, most preferably 40 μ M;

preferably, the photoreactive dye is PMA or EMA; preferably PMA.

5. The method according to any one of claims 1 to 4, which comprises a step of adding SDS to the cells before the cells are treated with the photoreactive dye;

preferably, the final concentration of SDS after addition is from 50 to 150ppm, more preferably from 90 to 110ppm, more preferably 100 ppm.

6. The method according to any one of claims 1 to 5, wherein the step of extracting the bacterial DNA is a step of extracting DNA by a cetyltrimethylammonium bromide method.

7. The method according to any one of claims 1 to 6, wherein the method is used for detecting the viable count of Escherichia coli in milk or drinking water.

8. The method of any one of claims 1 to 7, further comprising a dairy pretreatment step, wherein the pretreatment step is enriching Escherichia coli in milk;

preferably, the dairy product pretreatment step comprises centrifuging the dairy product, removing upper fat and supernatant, washing the precipitate, and then resuspending the precipitate.

9. The method according to any one of claims 1 to 8, further comprising the step of preparing a standard curve of the number of bacteria and the qPCR Cq value using a gradient diluted E.coli strain solution of known concentration;

preferably, the preparation step of the standard curve comprises: taking the viable bacteria suspension of the escherichia coli, diluting to ensure that the concentration of the bacteria liquid is 10⁷CFU/m L, then extracting DNA, and weighing the extracted DNA by 10 timesThe gradient was serially diluted 5 times, qPCR reactions were performed on DNA at each concentration, and a standard curve was calculated using CFX Manager 3.1.

10. The method according to any one of claims 1 to 9, wherein the step of removing the unreacted photoreactive dye comprises centrifuging the mixture of cells after the photoreaction and washing the precipitate.