CN105296668B

CN105296668B - Primer, probe and kit for specifically detecting type 3 ungulate bocavirus parvovirus

Info

Publication number: CN105296668B
Application number: CN201510723942.7A
Authority: CN
Inventors: 周宇; 唐连飞; 徐鹏; 宋斯伟; 朱事康; 于飞; 吕飞; 佟铁铸; 李春萍; 刘星; 刘中勇
Original assignee: HUIZHOU ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU OF PRC
Current assignee: HUIZHOU ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU OF PRC
Priority date: 2015-10-31
Filing date: 2015-10-31
Publication date: 2019-12-17
Anticipated expiration: 2035-10-31
Also published as: CN105296668A

Abstract

The invention discloses a primer, a probe and a kit for specifically detecting 3-type ungulate bocavirus. The detection kit and the detection reagent have the advantages of accurate detection, high sensitivity, strong specificity, simplicity, convenience and rapidness, and have good detection capability.

Description

Primer, probe and kit for specifically detecting type 3 ungulate bocavirus parvovirus

Technical Field

The invention relates to a biological detection technology, in particular to a primer, a probe and a kit for specifically detecting type 3 ungulate bocavirus parvovirus.

Background

The Porcine parvovirus (Bocavirus) belongs to the subfamily parvoviridae of the parvoviridae, including the subfamily uroviridae of the concentrated virus family, which mainly infects insects, and the subfamily uroviridae, which mainly infects vertebrates, the subfamily parvoviridae of the parvoviridae, which is divided into 6 genera including the genus parvovirus, Bocavirus, virus-dependent genus, mink aleppo virus, which belongs to the genus Bocavirus, is a single-stranded non-enveloped DNA virus, the genome of which is about 635.2 kb, which includes 3 Open Reading Frames (ORFs), which are NS1, NP1 and V P1/VP2. the respiratory system and the digestive system of the main invading pig, the positive rate of the Porcine PBoV of the pig herd pig group such as 30% ~, the Porcine high fever system, piglet diarrhea, etc. is 30% ~% based on the Porcine weaning syndrome of the Porcine reproductive system, the Porcine diarrhea, the Porcine reproductive system, the Porcine reproductive and the Porcine reproductive system, the Porcine reproductive and respiratory system, the Porcine reproductive system, the Porcine.

disclosure of Invention

In view of the above, the invention discloses a primer pair capable of accurately and rapidly detecting the ungulate bocavirus type 3.

Another object of the present invention is to provide a fluorescent probe used in combination with the primer set.

the third purpose of the invention is to provide a detection reagent, a kit and a reaction system thereof for detecting the ungulate bocavirus type 3.

The first purpose of the invention is realized by the following technical scheme: a PCR primer pair for detecting the ungulate bocavirus type 3 has the upstream primer with the nucleotide sequence shown in SEQ ID No.1 and the downstream primer with the nucleotide sequence shown in SEQ ID No. 2.

The second purpose of the invention is realized by the following technical scheme: a fluorescent probe matched with the primer pair for use, wherein the sequence of the fluorescent probe is PboVP1 shown as EQ ID No. 3; the 5 'end of the probe is marked with a fluorescent reporter group, and the 3' end of the probe is marked with a non-luminous fluorescent quenching group.

The fluorescent reporter group is any one selected from FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY 5; the quenching group, using the Black hole quencher BHQ1 from Biosearch Technologies, was very effective for dual-label probes, molecular beacons and FRET probes, and had excellent spectral overlap effect for all dyes in the green to black emission spectra.

The invention obtains all homologous gene sequences with 3-type ungulate bocavirus from NCBI database, screens out a highly conserved sequence (shown as SEQ ID NO. 4) from 1538 to 1671 of the entire sequence (GeneBank number: HQ 223038.1) of the 3-type ungulate bocavirus in NS1 gene by adopting MegAlign software, and designs primers and probes by using primer Express5.0 software by taking the highly conserved sequence as a template.

the primer pair for detecting UboV3 is based on NS1 gene of UboV3, adopts primer design software well known in the field to design primers, and obtains a detection primer and a probe with strong specificity for detecting UboV3 type through a large amount of sequence analysis, comparison and multiple tests. As will be appreciated by those skilled in the art, the NS1 gene has low homology, between 22.6-53.2%, among the various genotypes of porcine bocavirus, although the sequence homology within UboV3 is high, between 98.2-98.6%; however, it is difficult to design highly sensitive and specific diagnostic primers and probes due to the presence of multiple point mutations. After the inventors screened a highly conserved and unique fragment in the NS1 gene of UboV3 by MegAlign software, they repeatedly demonstrated and screened to finally determine a sequence of 134 base pairs (as shown in SEQ ID NO. 4). This fragment has a very high degree of conservation in the ungulate bocavirus type 3 (100% homologous fragments can be found in all homologous genomes), and can accurately distinguish the ungulate bocavirus type 3 from other porcine bocaviruses. It should be emphasized that the inventors found that this fragment does not have the phenomenon of gene insertion commonly existing in porcine bocavirus, and at the same time, in a specific reaction system, it has very high stability, and can keep the sequence consistent and few mutations after multiple rounds of amplification, which is very important for improving the accuracy of the diagnosis result and the diagnosis efficiency. The effect is obviously improved compared with the problem that the porcine bocavirus cannot be specifically amplified for diagnosis in the prior art, and no report of any document exists, and the document does not mention the conservation of the gene.

After determining the target fragment to be specifically amplified, the inventors performed a long-term screening based on evaluation by various bioinformatic software. In the prior art, as the target sequence of the diagnostic process cannot be determined, degenerate primers are designed to enhance the application universality, but primers with high degeneracy have influence on the amplification specificity. In addition, after the inventors have studied the target sequence aimed by the present invention, it is found that the content of t or c in the specific primer is too high, which is very likely to cause the stability of the target sequence to be decreased. Therefore, the t or c content of the specific primer in the present invention is strictly controlled to reduce its influence on the stability of the target sequence. This outstanding technical advance is fully reflected in the high reproducibility of the reaction system of the kit provided by the invention.

As mentioned above, the target sequence can only maintain its characteristic stability in a specific reaction system. Therefore, in order to achieve the objective of the present invention, the inventors have also conducted continuous search for the reaction conditions and reaction system of real-time fluorescence quantitative PCR in order to obtain the optimal reagent concentration and reaction temperature. The aspect of a reaction system is as follows: for Mg²⁺The concentration, the primer use concentration and the probe use concentration are repeatedly tested, and the related reagents are diluted in a multiple ratio to obtain the optimal use concentration in the system. Such as Mg²⁺Has important function for stabilizing base pairing and maintaining the reaction activity of polymerase, has similar influence mode to LAMP on PCR, and ensures that the reaction cannot be carried out when the concentration is too high or too low or the reaction time is prolonged and Mg in the reaction system is separated out due to the precipitation of magnesium pyrophosphate precipitate in the reaction process²⁺The concentration of (c) is continuously decreased. Especially Mg²⁺Too low a concentration, the stability of the target sequence will continue to decrease, giving diversity to the product sequences obtained after multiple cycles. And Mg²⁺Too high a concentration of (A) will result in a loss of the specific amplification effect of the primer and a failure to amplify the target sequence. Therefore, groping for Mg2⁺The optimum concentration is significant. After multiple experimental screening, the 20. mu.L PCR reaction system of the present invention preferably contains 1.0. mu.L of Mg with a concentration of 50mmol/L²⁺And (3) solution. In addition, the probe concentration, the amount of dNTP used, the template concentration, etc. all affect the performance of the primer and probe of the present invention, and long-term experimental verification is often required to find the optimum amount. In view of the specificity of the target sequence aimed at by the present invention, the simple application of the prior art cannot realize accurate and efficient diagnosis of the sample. In terms of reaction conditions, the reaction temperature and time in a similar method are referred to and adjusted for a plurality of times to determine appropriate reaction conditions. And in the experimental process, searching and verifying repeatability, sensitivity and specificity. Once the reaction system is changed, unknown mutation of the amplified target fragment may occur, resulting in false negative detection results.

The invention also provides application of the primer pair and the fluorescent probe in preparation of a detection kit or a diagnosis kit for the type 3 ungulate bocavirus.

The reaction system of the kit is as follows: the specific configuration of 20. mu.L reaction system was 2. mu.L of 10 XPCR reaction buffer, 1. mu.L of 50mM magnesium ion, 0.5. mu.L of 10mM dNTPs mixed solution, 10. mu.M PboVF10.5. mu.L, 10. mu.M PboVR10.5. mu.L, 10. mu.M PboV P10.5. mu.L, 5 u/. mu.L of Taq enzyme 0.2. mu.L, 10 to 50ng of test sample DNA template 1ML and the balance of pure water

Furthermore, the working condition of the reagent box in the application is 95 ℃ and 3 min; 95 ℃ for 5 s; 40 cycles of 55 ℃ 15s, 72 ℃ 40s were performed and then terminated.

When the kit is used, when the probe is complete, the fluorescent energy emitted by the 5 'end reporter gene is absorbed by the 3' end quenching group, and an instrument cannot detect a fluorescent signal; with the real-time fluorescent quantitative PCR, when Taq enzyme meets a probe combined with a template in the chain extension process, the probe is cut off by 5 '→ 3' exonuclease activity of the Taq enzyme, a report group is dissociated from the probe, the dissociated report group is far away from a quenching group, and the excited energy cannot be absorbed, so that the emitted fluorescent signal is detected by an instrument, each cycle receives collected data, the fluorescent signal has a synchronous exponential growth process along with the extension of a target fragment every time a PCR cycle is passed, and the result is judged according to an amplification curve after the reaction is finished, so that the detection and the quantification of the 3-type ungulate boca parvovirus are realized.

Compared with the prior art, the invention has the following advantages: the probe designed by the invention has good specificity and high sensitivity. The prepared kit is used for detecting UboV3, and has high sensitivity, high accuracy and strong specificity; the detection method can quickly detect whether the sample contains the UboV3 and can determine the copy number content of the UboV3 in the sample.

1. The high specificity is that the total DNA of the sample is taken as a template by adopting the PCR reaction system provided by the invention, other DNA viruses which are commonly present in live pigs are taken as a contrast, the fluorescence intensity change is detected through real-time fluorescence quantitative PCR, the result shows that the obvious fluorescence intensity change and good positive amplification result can be observed by taking the DNA extracted from the positive sample as the template through the real-time fluorescence quantitative PCR detection, and the fluorescence intensity of the DNA template extracted from other viruses is not changed.

2. The sensitivity is high: the amplification template can reach 1.44 copies/mu L, is 1 to 3 orders of magnitude higher than that of a conventional PCR method, can accurately detect the 3-type ungulate bocavirus parvovirus in a sample, is favorable for early diagnosis of diseases and takes related control measures to control the spread of epidemic diseases.

3. The reaction time is short, and the test efficiency is high: the cycle of each cycle in the present invention is 40S, which is lower than the reaction cycle of general PCR, thereby promoting the improvement of test efficiency.

4. The reproducibility is good: in the invention, the most conservative sequence containing 134 base pairs is screened out from the whole gene sequence of the 3-type ungulate bocavirus, and a special amplification primer and a probe are designed aiming at the sequence; under the reaction system provided by the invention, the target gene can be amplified with high fidelity and rarely undergoes point mutation, and the purpose of diagnosing the 3-type ungulate bocavirus parvovirus is finally realized; due to the stability of the system and the high conservation of the target gene, the invention has better reproducibility and has the advantage of industrial application.

Drawings

FIG. 1 is a standard curve for real-time fluorescent quantitative PCR detection of type 3 ungulate bocavirus parvovirus in example 3; the positive standard curve shows that the real-time fluorescent quantitative PCR is 1.44 multiplied by 10 for the porcine bocavirus²Copy/. mu.L-1.44X 10⁶The copy/. mu.L range has good linear relation, and the correlation coefficient is 0.997.

FIG. 2 shows the amplification results of the bovine parvovirus type 3 positive plasmid in example 3;

Degree of dilution	10⁸	10⁷	10⁶	10⁵	10⁴
						Ct value	27.09	24.37	20.65	17.79	14.74

FIG. 3 shows the specific detection results of the type 3 ungulate bocavirus parvovirus in example 4,

Wherein, 1 is the real-time fluorescent quantitative PCR amplification result of the 3-type ungulate bocavirus parvovirus prepared in the laboratory, 3 is the real-time fluorescent quantitative PCR amplification result of the porcine pseudorabies virus, 4 is the real-time fluorescent quantitative PCR amplification result of the porcine circovirus 2, 5 is the real-time fluorescent quantitative PCR amplification result of the classical swine fever virus, 6 is the real-time fluorescent quantitative PCR amplification result of the porcine reproductive and respiratory syndrome virus, 7 is the amplification result of the 2-type ungulate bocavirus parvovirus, and 8 is the amplification result of other porcine bocavirus subtypes (KM 402137, KM402138, KJ 755666) obtained in the laboratory;

FIG. 4 is a graph showing the sensitivity of real-time fluorescent quantitative PCR for detecting type 3 ungulate bocavirus parvovirus in example 5;

The positive plasmid containing the NS1 gene of the ungulate bocavirus type 3 obtained in the laboratory is sequentially diluted by 10 times, and the plasmid concentration represented by the curve marked by the curve number 9-15 in the figure is 1.44 copies/. mu.L-1.44 multiplied by 10⁶Copies/. mu.L.

FIG. 5 is a positive plasmid repeat test for real-time fluorescent quantitative PCR detection of the NS1 gene of the porcine bocavirus type 3 in example 6.

Detailed Description

In order to facilitate understanding for those skilled in the art, the present invention will be described in further detail below with reference to examples:

EXAMPLE 1 design and Synthesis of primers and probes

All the whole gene sequences belonging to the group of the ungulate bocavirus type 3 were downloaded from GenBank, and the near-whole gene sequence (KJ 755665) obtained in this laboratory was subjected to homology comparison using DNAStar software, and primers and TaqMan probes were designed using primer express5.0 software, and the primers and probes were synthesized by shanghai bio-chemical company. The length of the amplified target fragment is 134 bp.

The nucleotide sequence of the upstream primer is shown as PboVF 1;

The nucleotide sequence of the downstream primer is shown as PBoVR 1;

the nucleotide sequence of the probe used in combination with the above primer is shown in PboVP 1. The 5 'end of the probe is marked with a report FAM fluorescent dye, and the other 3' end is marked with a BHQ1 quenching group.

example 2 extraction of total DNA the following steps were performed:

Adding 2-4 ML PBS or physiological saline into the fecal sample, homogenizing, centrifuging at 12000r/min for 5min, collecting the supernatant, pulverizing the tissue sample, adding 2ML/g PBS, homogenizing, freeze thawing for 3-5 times, centrifuging at 12000r/min for 5min, collecting the supernatant, and performing the following tests. Extracting virus DNA (the DNA extracted in the test is only based on the DP315 kit, and other commercial virus DNA extraction kits are all suitable) according to the use instruction of a virus genome DNA/RNA extraction kit (DP 315) of Beijing Tiangen Biotechnology company, directly using or storing in a refrigerator at the temperature of minus 20 ℃ for later use, and storing in a refrigerator at the temperature of minus 70 ℃ for long time.

example 3 establishment of real-time fluorescent quantitative PCR amplification method

1. real-time fluorescent quantitative PCR reaction system

taking total DNA as a template to carry out real-time fluorescence quantitative PCR reaction, namely, 20 mu L of reaction system contains: 2.0. mu.L of 10 XPCR buffer, 2.0. mu.L of dNTPs (2.5mmol/L), 0.5. mu.L of primer PboVF1 (10. mu.l/L), 0.5. mu.L of primer PBoVR1 (10. mu.l/L), 1.0. mu.L of magnesium ions (50mmol/L), 0.5. mu.L of fluorescent probe (10. mu.l/L), 1.0. mu.L (10 ng-50 ng/. mu.L) of DNA template, 0.2. mu.L of Taq enzyme (5U).

2. Real-time fluorescent quantitative PCR reaction condition

After placing the sample tube into a 7500 fluorescence PCR instrument manufactured by ABI company, the following conditions were set: at 95 ℃ for 3 min; 95 ℃ for 5 s; 40 cycles of 55 ℃ and 15s, 72 ℃ and 40 s. Data were collected after each cycle was completed. And judging the result according to the amplification curve and the standard curve after the reaction is finished.

3. Establishing a real-time fluorescent quantitative PCR standard curve, cloning NS1 gene fragment of the hoof bocavirus type 3 into a pUCM-T vector to construct a positive plasmid, carrying out small extraction on the plasmid, and determining the content of the plasmid to be about 1.44 multiplied by 10¹⁰Copies/. mu.L. Then, the positive plasmid with 10 times of serial dilution is taken as a quantitative positive standard template to establish the real-time fluorescent quantitative PCR reaction of the 3-type ungulate bocavirus. The results are shown at 1.44X 10¹～1.44×10⁵The standard curve of the detection of the 3-type ungulate bocavirus parvovirus in the copy range has good linear relation (figure 1), and the detection of a plurality of positive recombinant plasmids has good amplification curve (figure 2).

EXAMPLE 43 specificity test of real-time fluorescent quantitative PCR method for ungulate bocavirus parvovirus

DNA extracted from positive plasmid is used as positive quality control contrast to detect positive samples of type 3 ungulate bocavirus, porcine pseudorabies virus, porcine circovirus, classical swine fever virus and porcine reproductive and respiratory syndrome virus and other subtype strains (KM 402137, KM402138 and KJ 755666) obtained in the laboratory.

The change in fluorescence intensity was detected by real-time fluorescent quantitative PCR. The result shows that the DNA extracted from the positive recombinant plasmid is taken as a template, obvious fluorescence intensity change and good positive amplification result can be observed through real-time fluorescent quantitative PCR detection, and the 3-type ungulate bocavirus has a positive amplification curve; the fluorescence intensity of the DNA template extracted from the porcine pseudorabies virus, the porcine circovirus, the classical swine fever virus and the porcine reproductive and respiratory syndrome virus and the DNA template of other subtype strains obtained in the laboratory is not changed, and an amplification curve is not generated. The real-time fluorescent quantitative PCR system established by the invention is prompted to have good specificity to the type 3 ungulate bocavirus parvovirus (figure 3).

EXAMPLE 5 sensitivity test

And (3) carrying out DEPC treatment on double distilled water to dilute the bovine parvovirus type 3 positive plasmid of the ungulate, and detecting the change of fluorescence intensity by real-time fluorescence quantitative PCR. The experimental result shows that the real-time fluorescence quantitative PCR detection can observe obvious fluorescence intensity change and a good positive amplification result, and the real-time fluorescence quantitative PCR kinetic curve shows that the reaction system with the concentration of more than 1.44 copies/muL has obvious fluorescence increase, but the reaction system with the concentration of less than 1.44 copies/muL and the negative control have no fluorescence increase, so the minimum detection limit of the real-time fluorescence quantitative PCR detection system is 1.44 copies, namely the sensitivity of the real-time fluorescence quantitative PCR detection system is 1.44 copies/muL (figure 4).

Example 6 repeatability test

The same dilution of the above-mentioned PoBV3 positive plasmid was used as template, and diluted in 10-fold gradient to 4 different gradient templates, and the reaction was repeated. And taking plasmid and negative serum diluent, extracting nucleic acid, repeating porous synchronous detection, and inspecting repeatability. (for concrete procedures, refer to Andre I, Hamel M. Rapid detection of porcine viral using RNA extracted direct Irom assisted PCR assay and one reverse PCR assay [ J ]. J Clinical Mi microbial 1995.33(2):287 and 291. Dungbo et al, establishment of porcine bocavirus real-time fluorescence quantitative PCR assay [ J ]. animal medical Advance, 2012,33(9): 70-74). The results are shown in Table 1.

TABLE 1 analysis of repeatability tests

	1：100	1：1000	1：10000	1：1000000
					Detection rate	100%	100%	100%	100%
Coefficient of variation CV	0.06%	1.04%	1.49%	1.03%

The foregoing is a detailed description of the invention, which is described in greater detail and not intended to limit the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications are possible without departing from the inventive concept, and such obvious alternatives fall within the scope of the invention.

SEQUENCE LISTING

<110> Huizhou entry and exit inspection and quarantine bureau of the people's republic of China

<120> primers, probes and kit for specifically detecting type 3 ungulate bocavirus

<130> 2015

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> DNA

<213> PboVF1

<400> 1

TGGGTGGAAC CAGCAAAA 18

<210> 2

<211> 20

<212> DNA

<213> PBoVR1

<400> 2

CCTCCAACGC ATTCGTAGAT 20

<210> 3

<211> 27

<212> DNA

<213> PboVP1

<400> 3

ACTCACAGCT CCTTCCACAA ACACCGT 27

<210> 4

<211> 134

<212> DNA

<213> Ungulate bocavirus type 3 (Unsulte Bocaparvovirus 3)

<400> 4

tgggtggaac cagcaaaatg tgtgcttgga ggaacaactg tgagagtcga tagaaaacac 60

aaagactcac agctccttcc acaaacaccg tgcataattt caacaaacaa caacatctac 120

gaatgcgttg gagg 134

Claims

1. A combination of a primer pair and a fluorescent probe for specifically detecting 3-type ungulate bocavirus parvovirus comprises an upstream primer and a downstream primer, and is characterized in that the nucleotide sequence of the upstream primer PboVF1 is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer PBoVR1 is shown as SEQ ID No. 2; the sequence of the probe PboVP1 is shown as SEQ ID NO. 3; the 5 'end of the probe is marked with a reporter group, and the 3' end of the probe is marked with a fluorescence quenching group; the fluorescence reporter group is any one of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5, and the quencher group is BHQ 1.

2. A real-time fluorescent quantitative detection kit for specifically detecting type 3 ungulate bocavirus, comprising the combination of the primer pair of claim 1 and a fluorescent probe.

3. The kit according to claim 2, wherein the reaction system of the kit is: the specific configuration of the 20. mu.L reaction system was 2. mu.L of 10 XPCR reaction buffer, 1. mu.L of 50mM magnesium ion, 0.5. mu.L of 10mM dNTPs mixed solution, 10. mu.M PboVF10.5. mu.L, 10. mu.M PboVR10.5. mu.L, 10. mu.M PboV P10.5. mu.L, 5 u/. mu.L of Taq enzyme 0.2. mu.L, 10ng-50 ng/. mu.L of the DNA template of the sample to be tested, and the balance of pure water.

4. The kit of claim 2, wherein the fluorescent quantitative PCR amplification conditions of the kit are as follows: at 95 ℃ for 3 min; 95 ℃ for 5 s; 40 cycles of 55 ℃ 15s, 72 ℃ 40s were performed and then terminated.