CN114134253A

CN114134253A - Fluorescent quantitative detection primer and kit for identifying African swine fever wild strain and gene deletion strain

Info

Publication number: CN114134253A
Application number: CN202111324516.8A
Authority: CN
Inventors: 陈耀; 曾繁聪; 黄淑坚; 马春全; 姜含雨; 辛宁; 柯骏鸿; 罗瑞; 谢梓民; 杨惠湖; 肖娅琪
Original assignee: Foshan University
Current assignee: Foshan University
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2022-03-04

Abstract

The invention aims to provide a fluorescent quantitative detection primer and a kit for identifying a wild strain and a gene deletion strain of African swine fever, and the primer and the kit comprise the primer, a probe and the kit containing the primer and the probe. The primer, the probe and the kit all aim at the I177L gene, a fluorescent quantitative TaqMan probe method can be established, and the minimum detection amount is 4.63 multiplied by 10¹copies/mu L, high sensitivity, good specificity, and the variation coefficient in batch and between batches is less than 2.2%. Therefore, the invention can be used as a rapid, sensitive and accurate detection means to be applied to actual production and epidemic situation monitoring, and provides reference for identifying and detecting infection of vaccine strains and wild strains in the future.

Description

Fluorescent quantitative detection primer and kit for identifying African swine fever wild strain and gene deletion strain

Technical Field

The invention relates to the technical field of gene detection, in particular to a fluorescent quantitative detection primer and a kit for identifying African swine fever wild strains and gene deletion strains.

Background

African Swine Fever Virus (ASFV) is a linear double-stranded DNA virus with a particle structure similar to that of iridovirus and a similar size, but the gene structure and replication pattern of the virus are different from those of iridovirus and have the same characteristics as poxviruses. In the classification of viruses, the international committee for classification of viruses classified iridoviridae and poxviridae in the fourth and fifth reports. With the development of molecular biology technology, DNA sequence analysis of african swine fever virus shows that although having some characteristics of poxvirus and iridovirus, african swine fever virus does not belong to any family previously defined by the international committee for virus classification, and is a completely new family of viruses, so that the virus is currently classified as african swine fever virus (Asfarviridae) african swine fever virus (Asfivims) and is the only member thereof, and is also the only DNA virus in arbovirus.

At present, the detection technologies for ASFV mainly include virus separation, red blood cell adsorption assay (HAD), ELISA, PCR, real-time fluorescent quantitative PCR, and the like.

The virus separation and the erythrocyte adsorption test are carried out in laboratories with 3 or more levels of biosafety, and the virus separation is combined with technologies such as HAD (Had-human immunodeficiency Virus) or PCR (polymerase chain reaction), ELISA (enzyme-linked immunosorbent assay) and the like to determine whether ASFV infection exists. In HAD, some non-erythrocyte-adsorbed strains cannot be detected by this method. The virus separation and HAD take long time, the technical requirement is high, the site requirement is high, and other detection technologies are required to assist whether ASFV infection exists or not during detection.

ELISA is the first choice serological detection method recommended by OIE, and most of the ELISA is an indirect detection method established by taking ASFV structural protein with good antigenicity as a coating antigen. The method has the advantages of low cost, good specificity, high sensitivity, high detection speed and the like, can be used for automatic detection of a large number of samples, and is the most widely applied detection method at present. However, compared with PCR, the method takes long time, and false positive is easy to appear in the result; the accuracy of the detection result is affected by improper sample preservation, such as high temperature or long sample preservation time.

Conventional PCR is the most primitive technique for detection from the molecular level, with some accuracy, but with the advent of fluorescent quantitative PCR, the sensitivity of conventional PCR is significantly weaker than both. The primer is designed by the Muero et al according to the p72 gene conserved sequence, the PCR method for rapidly detecting ASFV is established, and OIE authentication is obtained by the method. However, the research reports show that the method has a false negative result in detection, and the analysis and the finding are caused by the mismatching of the primer and the target gene sequence of part of genotype ASFV strains (gene II strains such as Georgia 2007/1 strains). In addition, one study showed that this PCR method gave false positive results in clinical sample testing.

The real-time fluorescent quantitative PCR technology is a method of adding fluorescent groups into a PCR reaction system, utilizing fluorescent signal accumulation to monitor the whole PCR process in real time, and finally carrying out quantitative analysis on an unknown template through a standard curve. The real-time fluorescent PCR effectively solves the problem that the conventional PCR can only be monitored at the end point, and further optimizes the PCR technology.

The fluorescent quantitative TaqMan probe method is a quantitative PCR technique with high specificity. The working principle of the kit is that a pair of PCR primers and a probe exist in a PCR reaction system, a 5 'end of the probe is marked with a reporter group, a 3' end of the probe is marked with a fluorescence quenching group, the probe is only specifically combined with a template, and the combining site of the probe is between the two primers. When the probe is complete, the fluorescence energy of the reporter group is absorbed by the quencher group, so the signal can not be collected by the instrument, as the reaction progresses, the Taq enzyme meets the probe, the probe is cut off by the activity of 3 '→ 5' exonuclease, so the fluorescence energy of the reporter group can not be absorbed by the quencher group, a fluorescence signal is generated, and the intensity of the signal represents the copy number of the template DNA.

The fluorescent quantitative dye method can be combined with all the dsDNA double helix minor groove areas. Since the fluorescence emitted in the free state is weak, but when it is bound to the double-stranded DNA, the fluorescence is greatly enhanced and the increase of the fluorescence signal is completely synchronized with the increase of the PCR product. The method has the advantages that the method can monitor the amplification of any dsDNA sequence, the detection method is simpler and has lower cost, but because the fluorescent dye can be combined with any dsDNA, the denaturation temperature of nonspecific products and primer dimers is lower than that of target products, and signals can be collected by a software analysis instrument in the melting curve reaction process for identification.

In summary, the real-time fluorescent quantitative PCR has the characteristics of strong specificity and high sensitivity, and is widely applied to detection of ASFV. At present, a plurality of fluorescence quantitative TaqMan probe methods aiming at ASFV exist, but a part of detection methods are not suitable for development of a gene deletion vaccine strain.

Disclosure of Invention

The invention aims to provide a fluorescent quantitative detection primer and a kit for identifying a wild strain and a gene deletion strain of African swine fever, so as to solve one or more technical problems in the prior art and provide at least one beneficial selection or creation condition.

The first purpose of the invention is to provide a primer for specifically amplifying an ASFV gene fragment, comprising:

an upstream primer fragment: 5'-TGGGCTTATTTTTGATTG-3' (SEQ ID NO. 1);

a downstream primer segment: 5'-ATTTGGTACCAGTAACACTA-3' (SEQ ID NO. 2).

The primer pair can specifically amplify a highly conserved gene fragment in the I177L gene of ASFV, and the size of the amplified target fragment is 167 bp.

A second object of the present invention is to provide a probe: 5'-GGACAACTTTGTCACCAGATG-3' (SEQ ID NO. 3).

Furthermore, the 5 'end of the probe fragment is connected with a fluorescent reporter group, and the 3' end of the probe fragment is connected with a quenching group. The fluorescence reporter group is selected from one of FAM, HEX, ROX or CY5, and the quenching group is selected from BHQ-1 or BHQ-2. By adding a fluorescent reporter group and a quencher group, the probe is specifically bound to the template only, and the binding site is between the two primers. When the probe is complete, the fluorescence energy of the reporter group is absorbed by the quencher group, so the signal can not be collected by the instrument, along with the progress of the reaction, Taq enzyme meets the probe, the probe is cut off by the activity of exonuclease from 3 'end to 5' end, so the fluorescence energy of the reporter group can not be absorbed by the quencher group, a fluorescence signal is generated, and the intensity of the signal represents the copy number of the template DNA.

The third purpose of the invention is to provide a kit, which comprises the primer and the probe.

Further, the kit also comprises a premixed Taq enzyme reagent with the concentration of 2 times, and specifically, the premixed Taq enzyme reagent with the concentration of 2 times is Premix Ex Taq. Premix Ex Taq is a special reagent for Real Time PCR (qPCR) reaction using the probe method. Is a 2X concentration Premix Type reagent, and the preparation of PCR reaction solution is very convenient and simple when experiments are carried out. When TliRNaseH (thermostable RNaseH) is added to Mix and PCR reaction is carried out using cDNA as a template, the inhibition of PCR reaction by residual mRNA in cDNA can be maximally suppressed. The improved DNA polymerase used in the HotStart method is used as the DNA polymerase in the product, so that nonspecific PCR amplification can be effectively inhibited, the PCR amplification efficiency is greatly improved, and high-sensitivity Real Time PCR amplification reaction is performed.

Further, the reaction system of the kit comprises 12.5 mu L of premixed Taq enzyme reagent with 2 times concentration and 8 mu L of ddH₂O, 0.5 muL of upstream primer segment, 0.5 muL of downstream primer segment, 1 muL of probe segment and 2 muL of template to be detected can form 25 muL of fluorescence quantitative PCR reaction bodyThe kit is used for qualitatively and quantitatively detecting ASFV in a sample.

Further, the concentration of the upstream primer segment, the downstream primer segment and the probe segment is 10 μ M. The reaction program is executed as follows: at 95 ℃ for 30 s; 95 ℃ for 5s, 60 ℃ for 30s, 40 cycles.

Further, the kit also comprises a plasmid standard substance, wherein the plasmid standard substance contains a target sequence fragment shown as SEQ ID NO. 4.

Further, the standard curve equation of the reaction system is-3.096 lgX + 36.18; coefficient of correlation R²0.998; the amplification efficiency E was 110.37%. The results show that there is a good linear relationship between standards of different concentrations, where the X-axis is the copy number of the plasmid standard and the Y-axis is the cycling threshold, consistent with the expected results.

The invention has the following beneficial effects:

at present, a plurality of fluorescence quantitative PCR methods aiming at ASFV are described in the literature, but some ASFV gene deletion vaccine strains are not suitable for development, and the diagnosis needs to be confirmed by combining a plurality of genes. The I177L gene is located in a middle stable gene region, has high sequence conservation, and can be used as a target gene for detection.

The invention establishes a fluorescent quantitative TaqMan probe method aiming at the I177L gene, and the minimum detected quantity is 4.63 multiplied by 10¹copies/mu L, high sensitivity, good specificity, and the variation coefficient in batch and between batches is less than 2.2%. Therefore, the invention can be used as a rapid, sensitive and accurate detection means to be applied to actual production and epidemic situation monitoring, and provides reference for identifying and detecting infection of vaccine strains and wild strains in the future.

Drawings

FIG. 1 is a standard graph of fluorescent quantitative PCR as described in example 3;

FIG. 2 is a graph of the fluorescent quantitative PCR sensitivity detection described in example 4;

FIG. 3 is a generic PCR sensitivity electrophoretogram described in example 4;

FIG. 4 is a graph of the fluorescent quantitative PCR-specific assay described in example 5.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1, synthesis and design of primers.

According to the ASFV sequence published on NCBI, the I177L gene of Georgia 2007/1 strain is picked, and the gene is completely consistent with the I177L gene of the domestic epidemic strain and is highly conserved. The whole gene was synthesized into a pUC-57 vector and named pUC-I177L. Designing a pair of specific primers and a probe of ASFV:

an upstream primer fragment: 5'-TGGGCTTATTTTTGATTG-3' (SEQ ID NO. 1);

a downstream primer segment: 5'-ATTTGGTACCAGTAACACTA-3' (SEQ ID NO. 2);

the probe fragment: 5 '-FAM-GGACAACTTTGTCACCAGATG-BHQ-3' (SEQ ID NO. 3).

Example 2, preparation of plasmid standards.

(1) PCR amplification and purification of the target fragment.

The target fragment was amplified using the I177L gene template using the forward and reverse primer fragment primers (10. mu.M) and a conventional PCR reaction system as shown in Table 1:

TABLE 1 conventional PCR reaction System

The reaction procedure is as follows: the reaction program is executed as follows: maintaining at 94 deg.C for 3 min; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 20 s; the temperature was 72 ℃ for 5 min.

After the PCR program is finished, all PCR products are added into 2% agarose gel containing Goldview, electrophoresis is carried out for 30min, a target band is cut off under the irradiation of an ultraviolet lamp, and gel recovery is carried out by using an Omega gel recovery kit. The method comprises the following specific steps:

(1-1) the single target DNA band was cut out of the agarose gel (excess was removed as much as possible), placed in a clean centrifuge tube, and weighed to calculate the gel weight (centrifuge tube weight was recorded in advance).

(1-2) to the gel block was added XP2 Binding Buffer in 1 volume (e.g. the gel weighed 100mg, the volume can be considered as 100. mu.L, and so on).

(1-3) bathing in a 50 ℃ water bath, wherein the centrifugal tube is gently turned upside down every 2-3 minutes until the sol solution is yellow to ensure that the gel block is fully dissolved. If the undissolved lumps still exist, some sol solution can be added or the mixture is kept standing for several minutes until the lumps are completely dissolved.

(1-4) transferring all the DNA/glue melt obtained in (1-3) into an adsorption column filled in a collecting tube, carrying out 13000rpm centrifugation for 1 minute, pouring off waste liquid in the collecting tube, and replacing the adsorption column into the collecting tube again.

(1-5) Add 300. mu.L of XP2 Binding Buffer to the column, centrifuge at 13000rpm for 1 minute, discard the collector tube and replace the adsorption column back in the collector tube.

(1-6) Add 700. mu.L of SPW Buffer to the adsorption column, centrifuge at 13000rpm for 1 minute, pour off the waste liquid in the collection tube, replace the adsorption column in the collection tube again.

(1-7) repeating the step (1-6) once.

(1-8) centrifuge at 13000rpm for 2 minutes, discard the collection tube, and load the adsorption column into a clean 1.5mL centrifuge tube.

(1-9) suspending and dropping 40 mu L of Elution Buffer to the middle position of the adsorption film, and standing for 2 minutes at room temperature. Centrifuge at 13000rpm for 1 minute. The DNA solution was collected. The DNA was stored at-20 ℃.

(2) And (4) connecting target fragments.

Vector ligation of the PCR-purified product obtained in step (1-9) was performed with reference to the instruction manual of Tarkara pMD18-T Vector, as shown in Table 2:

TABLE 2 ligation reaction System

After mixing, the mixture is put into a PCR instrument to act for 4h at 16 ℃ and stay overnight at 4 ℃ to obtain a ligation product.

(3) Screening and identifying recombinant plasmids.

(3-1) adding the ligation products obtained in step (2) to 50. mu.L of competent cells, respectively. Positive controls for the pMD18-T plasmid were set, as well as a competent cell control without any plasmid DNA added.

(3-2) after gently mixing the ligation product and competent cells, immediately ice-cooling for 30min, then heat-shocking for 90s at 42 ℃, and then immediately ice-cooling for 10 min.

(3-3) 400. mu.L of fresh LB liquid medium was added to each tube, and the mixture was incubated at 37 ℃ for 45min with slow shaking.

(3-4) spreading 100. mu.L of the bacterial liquid on 1.5% (W/V) LB agar plate containing Amp (100. mu.g/mL), and performing inverted culture at 37 ℃ for 12-16 h.

(4) Screening and identifying recombinant plasmids.

Picking a single colony which grows well in 500 mu L of LB liquid culture medium containing Ampicillin (AMP), placing the single colony in a constant temperature shaking table at 37 ℃ for shaking culture for 4-6 h, and taking a suspected positive bacterial liquid as a template for PCR identification, wherein the bacterial liquid is shown in Table 3:

TABLE 3 PCR reaction System

After the components are mixed evenly, a PCR program is executed: maintaining at 94 deg.C for 5 min; at 94 ℃ for 30s, at 55 ℃ for 30s, at 72 ℃ for 20s, for 35 cycles; the temperature was 72 ℃ for 5 min. And (4) sending the bacterial liquid which is identified as positive by PCR to biological engineering limited company for sequencing.

(5) And (4) extracting positive plasmids.

And (3) naming the bacterial liquid with correct sequencing result in the step (4) as 18T-I177L, carrying out amplification culture, and carrying out plasmid extraction by using a TaKaRa plasmid extraction kit, wherein the specific steps are as follows:

(5-1) cultivation of Escherichia coli. A single colony is selected from a plate culture medium and inoculated into 1-4 ml of liquid culture medium containing antibiotics, and the liquid culture medium is cultured at 37 ℃ overnight (the cells are difficult to crack after being cultured for 12-16 hours, and the yield of plasmids is reduced along with the cells cultured for more than 16 hours).

(5-2) 1-4 ml of overnight culture broth was centrifuged at 12000rpm for 2 minutes, and the supernatant was discarded.

(5-3) the bacterial pellet was suspended thoroughly with 250. mu.l of Solution I (containing RNase A).

(5-4) adding 250ul of Solution II, slightly turning and mixing up and down for 5-6 times to fully crack the thalli and form a transparent Solution.

(5-5) adding 350ul of 4 ℃ precooled Solution III, slightly turning and mixing up and down for 5-6 times until compact aggregate is formed, and then standing for 2 minutes at room temperature.

(5-6) centrifugation was carried out at 12000rpm for 10 minutes at room temperature, and the supernatant was collected.

(5-7) Spin Column in the kit was mounted on the Collection Tube.

(5-8) the supernatant of the operation (5-6) was transferred to a Spin Column, centrifuged at 12000rpm for 1 minute, and the filtrate was discarded.

(5-9) 500ul of Buffer WA WAs added to the Spin Column, centrifuged at 12000rpm for 30 seconds, and the filtrate WAs discarded.

(5-10) 700. mu.l of Buffer WB was added to Spin Column, centrifuged at 12000rpm for 30 seconds, and the filtrate was discarded.

(5-11) repeating the operation once (5-10).

(5-12) Spin Column was replaced on the Collection Tube and centrifuged at 12000rpm for 1 minute to remove the residual wash.

(5-13) the Spin Column was put on a new 1.5ml centrifuge tube, 50ul of sterilized water or Elution Buffer was added to the center of the Spin Column membrane, and left to stand at room temperature for 1 minute.

(5-14) DNA was eluted by centrifugation at 12000rpm for 1 minute.

The plasmid concentration was measured using a ultramicro fluorescence spectrophotometer and determined according to the gene copy number (copies/uL) ═ 6.02X 10²³X plasmid concentration (ng/uL). times.10^-9/[ plasmid size (bp) × 660]Calculating the copy of the geneThe shellfish number is then diluted to 100copies/uL by a 10-fold ratio and stored at-20 ℃ for later use.

Example 3 construction of fluorescent quantitative PCR reaction system and standard curve.

The 18T-I177L plasmid was diluted in a gradient to give 4.63X 10⁹copies/μL～4.63×10⁰copies/. mu.L 10 dilutions of standard, 4.63X 10⁹copies/μL～4.63×10²copies/. mu.L as reaction template, 3 replicates per dilution and a negative control was established. The reaction system is shown in table 4, and the result is shown in fig. 1, wherein the standard curve equation is that Y is-3.096 lgX + 36.18; coefficient of correlation R²0.998; the amplification efficiency E is 110.37%, and the results show that there is a good linear relationship between the standards at different concentrations, where the X-axis is the copy number of the plasmid standard and the Y-axis is the cycle threshold, which is consistent with the expected results.

TABLE 4 fluorescent quantitative PCR reaction System

The reaction program is executed as follows: at 95 ℃ for 30 s; 95 ℃ for 5s, 60 ℃ for 30s, 40 cycles.

Example 4, sensitivity assay.

Mixing 4.63 × 10⁰copies/μL～4.63×10⁷The plasmid standard samples of 8 dilutions were used as templates in copies/μ L, amplified according to the fluorescent quantitative PCR reaction system provided in example 3, and at the same time, the same templates were subjected to the ordinary PCR amplification, and the lowest detection limits of the real-time fluorescent quantitative PCR and the ordinary PCR were determined and the sensitivities of the two methods were compared. As shown in FIG. 2, the minimal amount of the positive standard detected by the fluorescent quantitative PCR reaction system provided in example 3 was 4.63X 10¹copies/. mu.L. As shown in FIG. 3, lane M is DL500 DNA Marker; lanes 1 to 10 are 4.63X 10⁹copies/μL～1.23×10⁰Samples of copies/. mu.L; lane 11 negative control. It can be seen that the minimum detectable amount of the ordinary PCR was 4.63X 10⁴copies/. mu.L. In contrast, example 3 establishes practiceCompared with the common PCR detection method, the time-fluorescence quantitative PCR detection method has the advantage that the sensitivity is higher by about 3 orders of magnitude.

Example 5, specificity test.

The specificity of the fluorescent quantitative PCR reaction system provided in example 3 was verified by amplifying the cDNA of Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the DNA of porcine circovirus type 2 (PCV2) and pseudorabies virus (PRV) using established real-time fluorescent quantitative PCR method. The results are shown in fig. 4, which indicates that the fluorescent quantitative PCR reaction system provided in example 3 has specific amplification only for ASFV, and has no fluorescent signal for CSFV, PRRSV, PCV2 and PRV, indicating that the reaction system has good specificity.

Example 6, repeatability tests.

3 batches of plasmids are respectively subjected to 3-batch repeated and 3-batch repeated real-time fluorescent quantitative PCR amplification, the change condition of the Ct value is compared, the stability of the method is verified, and the stability of the method is evaluated by using the batch and batch variation coefficients. The results are shown in Table 5, where the intra-batch coefficient of variation is less than 0.969%, the inter-batch coefficient of variation is less than 2.178%, and the reproducibility is good.

TABLE 5 fluorescent quantitative PCR repeatability test results

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

SEQUENCE LISTING

<110> institute of Buddha science and technology

<120> fluorescent quantitative detection primer and kit for identifying African swine fever wild strain and gene deletion strain

<130> 2021

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

<400> 1

tgggcttatt tttgattg 18

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

atttggtacc agtaacacta 20

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<400> 3

ggacaacttt gtcaccagat g 21

<210> 4

<211> 167

<212> DNA

<213> African Swine fever virus

<400> 4

tgggcttatt tttgattgtg ccaagcttca gccactaaca accaaatcaa atgtaacatc 60

tggtgacaaa gttgtccata ttggacaaac attcgaatat aataatcttt taatgtggaa 120

agttaatgat cagggctttt taaatattag tgttactggt accaaat 167

Claims

1. The primer is characterized by comprising an upstream primer segment shown as SEQ ID NO.1 and a downstream primer segment shown as SEQ ID NO. 2.

2. The probe is characterized by comprising a probe segment shown as SEQ ID NO. 3.

3. The probe of claim 2, wherein the probe fragment has a fluorescent reporter group attached to its 5 'end and a quencher group attached to its 3' end.

4. A kit comprising the primer combination of claim 1 and the probe of claim 2 or claim 3.

5. The kit of claim 4, further comprising 2-fold concentration of premixed Taq enzyme reagent.

6. The kit of claim 5, wherein the 2-fold concentration of premixed Taq enzyme reagent is Premix Ex Taq.

7. The kit of claim 5 or 6, wherein the reaction system of the kit comprises 12.5 μ L of premixed Taq enzyme reagent at 2-fold concentration, 8 μ L of ddH₂O, 0.5. mu.L of the upstream primer fragment, 0.5. mu.L of the downstream primer fragment, and 1. mu.L of the probe fragment.

8. The kit of claim 7, wherein the concentration of the forward primer fragment, the reverse primer fragment, and the probe fragment is 10 μ M.

9. The kit according to claim 7, further comprising a plasmid standard containing a sequence fragment of interest as shown in SEQ ID No. 4.

10. The kit of claim 7, wherein the reaction system has a standard curveThe equation is-3.096 lgX + 36.18; coefficient of correlation R²0.998; the amplification efficiency E was 110.37%.