Primer combination for simultaneously detecting eight herpes viruses through single tube, PCR (Polymerase Chain Reaction) detection system and application

Abstract

The invention discloses a primer combination for simultaneously detecting eight herpes viruses by a single tube, a PCR (Polymerase Chain Reaction) detection system and application, and relates to the technical field of herpes virus detection. Aiming at eight human herpes viruses, the invention provides a corresponding primer combination and an octuple PCR detection system, the detection sensitivity to single and mixed infection of the herpes viruses reaches 1 * 10 < 4 > copies (50 mu L system), non-specific amplification is avoided, and the eight human herpes viruses can be detected at the same time. When the system is used for detecting 30 cell samples, one EBV sample is detected to be positive, the rest EBV samples are detected to be negative, the result is consistent with the result obtained by using an EBV rapid visual detection system established in an earlier stage of a laboratory, and very high accuracy is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of herpes virus detection, in particular to a primer combination for simultaneously detecting eight kinds of herpes viruses in a single tube, a PCR detection system and application. BACKGROUND

[0002] Cell culture technology is a technology that simulates the in vivo environment to make cells grow and reproduce in vitro, which has developed from monolayer adherent culture to three-dimensional culture and is widely used in many fields. However, it faces the challenge of exogenous pollution, among which viral pollution is particularly tricky because it is difficult to detect and eradicate and has far-reaching effects, so it is very important to prevent its occurrence.

[0003] Early part of the cell lines may have been infected with viruses but were not discovered due to limitations of detection technology. Today, detection levels have improved, and cell line purity requirements are more stringent, and viral contamination needs to be excluded to ensure accurate experimental results. In the field of biological products, viral contamination detection is more critical. Biological products are mostly produced based on cell culture, and if contaminated, not only will it lead to production batch rejection and economic losses, but also may expose users to infection, immune abnormalities, and even life-threatening risks. Strict detection can control the quality from the source and ensure clinical safety

[0004] Human herpesviruses (HHVs) belong to the Herpesviridae family, which are a class of medium-sized, enveloped double-stranded DNA viruses. There are currently eight types of herpes viruses known to infect humans, which are divided into three subfamilies according to their biological characteristics, genomic sequences, and structural and physical-chemical properties: alpha herpesvirus subfamily (types 1-3), beta herpesvirus subfamily (types 5-7), and gamma herpesvirus subfamily (types 4 and 8). In cell culture, viral contamination detection is crucial and directly affects the reliability of experimental results and the safety of biological products. Currently, the main methods for detecting human herpesviruses in clinical settings include virus isolation and culture, serological detection, and nucleic acid detection.

[0005] Nucleic acid detection methods are currently the most commonly used methods, including polymerase chain reaction and isothermal amplification techniques. Polymerase chain reaction can be further divided into conventional PCR, real-time quantitative PCR (qPCR), digital PCR (dPCR), and multiplex PCR.

[0006] Multiple PCR can detect multiple viruses at the same time in a single reaction, which is suitable for the differential diagnosis of mixed infection samples, so as to accurately identify all related pathogens in time. However, multiple PCR still needs to overcome several technical challenges in practical application. First, the mutual interference between primers is a common problem of multiple PCR. Since multiple primers exist in the same reaction system at the same time, primer dimers are easily formed or cross-reactions occur, resulting in false positive problems. Second, the difference in amplification efficiency is also a big challenge for multiple PCR. During the reaction process, high-abundance targets tend to occupy reaction resources first, inhibiting the amplification of low-abundance targets, and affecting the accuracy of the detection results. Third, when performing ultra-multiple amplification, that is, simultaneously amplifying more than the conventional number of target sequences in one reaction, as the number of targets increases, the complexity of primer design also increases, and the competition effect in the reaction system is more obvious. Common herpes viruses include HSV-1, HSV-2, VZV (varicella-zoster virus), EBV, HCMV (human cytomegalovirus), HHV-6, HHV-7 and HHV-8, a total of 8 kinds. As the same virus category, these members are highly similar in genotype, have the characteristics of cross-infection and synergistic effect, but compared with the multiple PCR system for different types and different categories of viruses (such system is relatively easy to establish due to significant differences between viruses), the simultaneous detection system for the eight herpes viruses in the same category has not yet been achieved.

[0007] Therefore, in view of the relative ease of establishing a multiple PCR system for different types and different categories of viruses, and the many challenges faced by the simultaneous detection of the eight human herpes viruses in the same category, there is an urgent need to invent a detection method that can simultaneously detect them with high accuracy and high sensitivity, while being free from primer interference and cell matrix interference. SUMMARY

[0008] The present application provides a primer combination for simultaneously detecting eight herpes viruses in a single tube, a PCR detection system and application. The present application designs a corresponding primer combination for eight types (HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7 and HHV-8) of human herpes viruses. Under conventional experimental conditions, the simultaneous detection of eight human herpes viruses can be achieved by a single tube reaction, which is suitable for the scene of comprehensive screening of samples infected with human herpes viruses. The following technologies are used.

[0009] In a first aspect of the present application, a primer combination for simultaneously detecting eight human herpes viruses in a single tube is provided, which consists of forward primers and reverse primers of nucleotide sequences shown in SEQ ID NO. 9-SEQ ID NO. 24.

[0010] In a second aspect of the present application, an octaplex PCR detection system for simultaneously detecting eight human herpes viruses in a single tube is provided, comprising the primer combination.

[0011] Further, the plasmid standard for detecting HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7 and HHV-8 is also included.

[0012] Further, the plasmid standard for detecting HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7 and HHV-8 is also included.

[0013] In a third aspect of the present application, a product for simultaneously detecting eight human herpes viruses in a single tube is provided, comprising the primer combination, or any of the octaplex PCR detection systems.

[0014] In a fourth aspect of the present application, a method for simultaneously detecting eight human herpes viruses in a single tube is provided, which is not for the purpose of disease diagnosis and treatment, and the method comprises using the primer combination, or any of the octaplex PCR detection systems, or the product to simultaneously detect eight human herpes viruses in a sample to be tested.

[0015] The method for detecting eight human herpes viruses provided by the present application is mainly used for scientific research, and is not for disease diagnosis and treatment. Of course, the primer combination, the octaplex PCR detection system and the product provided by the present application can also be applied to disease diagnosis and treatment, and have higher sensitivity and higher accuracy.

[0016] Further, in the primer combination, the concentration of the forward primer and the reverse primer for detecting VZV is 150 nM, the concentration of the forward primer and the reverse primer for detecting HSV-1 and EBV is 300 nM, and the concentration of the forward primer and the reverse primer for detecting HSV-2, HCMV, HHV-6, HHV-7 and HHV-8 is 200 nM.

[0017] Further, when the primer combination is used for PCR amplification, the annealing temperature is 55-63℃. Further, the annealing temperature is 59℃.

[0018] Compared with the prior art, the present application has the advantages that the specific primer combination for simultaneously detecting eight human herpes viruses is designed, and the corresponding octaplex PCR detection system is established, and the detection sensitivity of the system for single and mixed infections of herpes viruses is 1×10 4Copy (50 μL system), no non-specific amplification, can detect 8 human herpes virus at the same time. Using this system to detect 30 cell samples, 1 case of EBV positive was detected, and the rest were negative. The results were consistent with the results of the EBV rapid visualization detection system established in the laboratory earlier, and the accuracy was very high. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The eight combinations of the five PCR detection systems are shown in the figure. Lanes 1 and 2 of each figure are parallel experimental groups. M: Marker DL2000; A: HHV-8 (437 bp) + HSV-1 (367 bp) + HCMV (257 bp) + HHV-6 (142 bp) + EBV (95 bp); B: HHV-8 (437 bp) + HSV-1 (367 bp) + HCMV (257 bp) + HSV-2 (159 bp) + EBV (95 bp); C: HSV-1 (453 bp) + HHV-6 (334 bp) + HCMV (257 bp) + HHV-8 (198 bp) + HSV-2 (124 bp); D: HSV-1 (367 bp) + HSV-2 (290 bp) + HHV-8 (198 bp) + HHV-6 (142 bp) + EBV (95 bp); E: HSV-2 (439 bp) + HSV-1 (367 bp) + HCMV (257 bp) + HHV-8 (198 bp) + HHV-6 (142 bp); F: HHV-8 (437 bp) + HSV-1 (367 bp) + HCMV (257 bp) + VZV (122 bp) + EBV (95 bp); G: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-8 (198 bp) + HSV-2 (124 bp); H: HSV-2 (439 bp) + HSV-1 (367 bp) + HCMV (257 bp) + HHV-8 (198 bp) + VZV (122 bp).

[0020] Figure 2 The specificity of the five pairs of primers of the five PCR detection systems is shown in the figure. Single primer refers to a primer corresponding to the virus; mixed template refers to a primer mixed with five virus positive templates, i.e. HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-8 (198 bp) + HSV-2 (124 bp).

[0021] Figure 3The amplification electrophoresis result of six-plex PCR detection system. Among them, the mixed primer refers to: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + EBV (95 bp); the mixed template is selected from the positive templates of six viruses.

[0022] Figure 4 The amplification electrophoresis result of seven-plex PCR detection system. In figure A, 1 represents: HSV-1 (453 bp) + HHV-6 (398 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + EBV (95 bp); 2 represents: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-6 (223 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + EBV (95 bp); 3 represents: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + HHV-6 (125 bp) + EBV (95 bp); in figure B, the mixed template represents the positive templates of seven viruses; the mixed primer is: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-6 (223 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + EBV (95 bp).

[0023] Figure 5 The amplification electrophoresis result of eight-plex PCR detection system. Among them, the mixed primer is: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-6 (223 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + HHV-7 (114 bp) + EBV (85 bp); the mixed template is the positive templates of eight viruses.

[0024] Figure 6 The sensitivity detection result of eight-plex PCR detection system for detecting eight herpes viruses. Among them, the mixed primer represents: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-6 (223 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + HHV-7 (114 bp) + EBV (85 bp).

[0025] Figure 7The detection results of the sensitivity of the octuple PCR detection system for detecting one herpes virus. The mixed primers represent: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-6 (223 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + HHV-7 (114 bp) + EBV (85 bp).

[0026] Figure 8 The specificity verification results of the octuple PCR detection system. In which, the A figure represents the single primer specificity verification with the DNA of B95-8 (EBV) cells as a template; the B figure represents the single primer specificity verification with the DNA of HUV-EC-C (HHV-6) cells as a template; the C figure represents the single primer specificity verification with the DNA of BCBL-1 (HHV-8) cells as a template; the mixed primers represent: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-6 (223 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + HHV-7 (114 bp) + EBV (85 bp).

[0027] Figure 9 The annealing temperature optimization verification results of the octuple PCR detection system.

[0028] Figure 10 The primer concentration optimization verification results of the octuple PCR detection system.

[0029] Figure 11 The amplification electrophoresis results of the octuple RPA detection system. In which, 1 represents that the primer concentrations of HSV-1, VZV, EBV and HSV-2 are all 100 nM; 2 represents that the primer concentrations of each virus are all 200 nM; 3 represents that the primer concentrations of each virus are all 300 nM; 4 represents that the primer concentrations of each virus are all 400 nM; 5 represents that the primer concentrations of each virus are all 500 nM.

[0030] Figure 12 The detection results of the octuple PCR detection system of the present application for detecting human herpes viruses in 30 cell strains. In which, the positive control represents: HSV-1 (453 bp) + VZV (326 bp) + HCMV (257 bp) + HHV-6 (223 bp) + HHV-8 (198 bp) + HSV-2 (159 bp) + HHV-7 (114 bp) + EBV (85 bp). DETAILED DESCRIPTION

[0031] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0032] 1. Experimental materials

[0033] In the following specific implementation cases provided by the present application, the E. coli strain DH5a and the pMD18-T vector are preserved and provided by the laboratory. All cell lines and live viruses of HSV-1, HSV-2, VZV, EBV and HCMV are preserved and provided by the China Center for Type Culture Collection (CCTCC) of Wuhan University. The cell lines used in the experiment are shown in Table 1 below.

[0034] Table 1 Cell lines used in the experiment

[0035]

[0036]

[0037] 2. Experimental reagents, consumables and instruments

[0038] The reagents, consumables and main experimental instruments required in the experiment are shown in Tables 2-4 below.

[0039] Table 2 Reagents, consumables and main experimental instruments required in the experiment

[0040]

[0041] Table 3

[0042]

[0043]

[0044] Table 4

[0045]

[0046] Example 1: Cell culture, recovery, subculture, cryopreservation, and DNA extraction

[0047] 1. Cell culture

[0048] In this embodiment, the cell culture is carried out according to the conventional experimental method summarized by the China Center for Type Culture Collection (CCTCC).

[0049] 2. Cell recovery

[0050] The cell sample is thawed, centrifuged, resuspended and cultured, and finally the culture bottle is placed in a 37℃, 5% CO2 incubator.

[0051] 3. DNA extraction

[0052] (1) Cell line DNA extraction

[0053] Select cells in good growth condition, and after trypsin digestion and centrifugal treatment, gently blow to form a cell suspension; according to the instruction of the blood / cell / tissue genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., extract the experimental operation. The collected cell DNA is ready for use, and is labeled in an EP tube.

[0054] For the convenience of subsequent experiments, the concentration of the extracted DNA can be determined using a NanoDrop microspectrophotometer, diluted with ddH2O to 50 ng / μL, and stored in a-20℃ refrigerator for standby.

[0055] (2) Virus DNA extraction

[0056] The virus liquid of HSV-1, HSV-2, VZV, EBV and HCMV preserved by the China Center for Type Culture Collection (CCTCC) is used to remove other impurities to obtain the DNA of the five viruses according to the operation instruction of the Viral DNA kit of OMEGA company, and is stored in a-20℃ refrigerator for standby.

[0057] Example 2: Design and screening of octupling PCR primer combination

[0058] 1. Octupling PCR detection method

[0059] (1) Primer design

[0060] In view of the high similarity of the 8 virus genotypes in the same herpes virus family, the present application establishes the core principle of "intra-species conservation, significant inter-species difference, and gradient distribution of fragment size" for primer design, which breaks through the technical bottleneck of confusion between cross-reaction and detection in conventional design.

[0061] To ensure the universality of the primers to different strains of the same virus, the present application retrieves multiple reference sequences of each virus (such as HSV-1 covering 10 strains of NC_001806.2, HSV-2 covering 8 strains of NC_001798.2) from the NCBI database, and uses ClustalX for sequence alignment to screen regions with no base variation for more than 200 bp as candidate targets. For example, the 453 bp fragment of HSV-1 is derived from the conserved region of the UL30 gene, and the sequence consistency in 15 clinical isolates is 99.2%; the 326 bp fragment of VZV is located in the ORF62 gene, and there is no base difference in 12 global epidemic strains, ensuring the wide coverage of the primers to the same virus.

[0062] In view of the high sequence homology of viruses in the same family, the BLAST whole genome alignment method is used to calculate the sequence similarity of the candidate target regions among the 8 viruses, and fragments with a similarity < 60% are strictly screened. Specifically, the designed primer sequence is aligned with the whole genome of the other 7 viruses through the Primer-BLAST tool to ensure that there is a continuous difference of ≥ 5 bases in the primer binding region between species. For example, the similarity of the 257 bp fragment of HCMV to the corresponding region of HSV-1 is only 42%, and the homology of the 198 bp fragment of HHV-8 to HHV-6 is less than 38%, which avoids cross amplification from the sequence level.

[0063] To achieve the clarity of electrophoretic separation, the size difference of the target fragments is strictly controlled during primer design: the length difference between adjacent fragments is ≥ 50 bp, and the overall length is distributed in steps from 85 bp to 453 bp (85 bp, 114 bp, 124 bp / 159 bp, 198 bp, 223 bp, 257 bp, 326 bp, 453 bp). For example, the fragment of HSV-2 is adjusted from the initial design of 124 bp to 159 bp, forming a 45 bp difference with HHV-7 of 114 bp; the fragment of EBV is optimized from 95 bp to 85 bp, maintaining a 29 bp interval with HHV-7 of 114 bp, ensuring that each band can be clearly distinguished in 3% agarose gel electrophoresis.

[0064] Finally, HSV-1 gene (NC_001806.2), HSV-2 gene (NC_001798.2), VZV gene (NC_001348.1), EBV gene (NC_001847.1), HCMV gene (NC_006273.2), HHV-6 gene (NC_001664.4), HHV-7 gene (NC_001716.2) and HHV-8 gene (NC_009333.1) are selected, 50 pairs of primers are designed, and multiple rounds of optimization design are carried out, and 33 pairs of primers with strong specificity are screened.

[0065] Among them, there are 5 primer pairs targeting the HSV-1 gene (NC_001806.2), 6 primer pairs targeting the HSV-2 gene (NC_001798.2), 5 primer pairs targeting the VZV gene (NC_001348.1), 3 primer pairs targeting the EBV gene (NC_001847.1), 2 primer pairs targeting the HCMV gene (NC_006273.2), 5 primer pairs targeting the HHV-6 gene (NC_001664.4), 3 primer pairs targeting the HHV-7 gene (NC_001716.2), and 4 primer pairs targeting the HHV-8 gene (NC_009333.1).

[0066] (2) Establishment of a five-plex PCR system

[0067] Five viruses were selected from the eight viruses. From the 33 primer pairs screened above, a pair of primers was selected for each of the five viruses, in equal proportions. During the primer selection process, the target band sizes corresponding to the primers were considered to ensure even separation of the five fragments. Ultimately, eight combinations were developed to attempt to construct a five-plex PCR detection system.

[0068] Each combination was amplified by adding a positive template for the corresponding virus. After consulting the literature, it was determined that BCBL-1 cells are positive for HHV-8, HUV-EC-C cells are positive for HHV-6, and B98-5 cells are positive for EBV. Therefore, when establishing the quintuple PCR system, the herpes virus-positive templates used were: four viral DNAs: HSV-1, HSV-2, VZV, and HCMV; total DNA from B98-5, HUV-EC-C, and BCBL-1 cells; and an HHV-7 standard plasmid. These herpes virus-positive templates were synthesized by Wuhan Tianyi Huiyuan Company.

[0069] The primer mix added to the quintuple PCR system consists of five selected primer pairs diluted to a 10 μM working solution and mixed in equal proportions. The specific amplification reaction system is shown in Table 5. After the reaction is completed, 10 μL of the product is directly analyzed by 3% agarose gel electrophoresis (voltage 130 V, current 220 A) without adding additional loading buffer to detect the amplified product and observe the final results.

[0070] The experimental results are as follows Figure 1 As shown in the figure. Of the eight experimental groups, only group G successfully amplified five target fragments; the remaining experimental groups only amplified three or four specific bands. Group G targeted HSV-1, VZV, HCMV, HHV-8, and HSV-2, with target fragment sizes of 453 bp, 326 bp, 257 bp, 198 bp, and 124 bp, respectively.

[0071] To further determine the specificity of the primer set, we added each pair of primers to the corresponding positive template for single PCR amplification. At the same time, we mixed the five pairs of primers in equal proportions to configure a mixed primer set, and added them to five corresponding single positive templates, respectively, and a mixed positive template of the five viruses for amplification.

[0072] The results of agarose gel electrophoresis are shown in Figure 2 It can be seen that the amplified fragments of the five viruses are of the expected size and have strong specificity; in the mixed primer set, even if only one virus exists in the system, it can be accurately detected, and the results are specific; when the five viruses are amplified at the same time, the corresponding target fragment size is consistent with the expected value. These results show that the five-plex PCR system can simultaneously detect HSV-1, VZV, HCMV, HHV-8 and HSV-2. Therefore, we selected this combination for subsequent experiments.

[0073] (3) Establishment of six-plex PCR system

[0074] Based on the above five-plex PCR reaction system, we added EBV primers to the five-plex PCR primer combination in proportion, and the corresponding amplified target fragment size was 95 bp. The total DNA of B95-8 cells was used as the positive template for amplification. The specific amplification reaction system is shown in Table 5.

[0075] After repeated attempts, we found that the HSV-2 target band amplification was abnormal, so we replaced the new HSV-2 primers, and the amplified target fragment size was 159 bp, thus establishing a six-plex PCR detection system, and the results are shown in Figure 3 , indicating that the six-plex PCR system can simultaneously detect HSV-1, VZV, HCMV, HHV-8, HSV-2 and EBV, and has strong specificity.

[0076] (4) Establishment of seven-plex PCR system

[0077] Based on the size distribution of the six target fragments in the above six-plex PCR reaction system, and referring to the principles and methods of the above primer design, we added three designed HHV-6 primers to the primer combination of the above six-plex PCR system, and the corresponding target fragment sizes were 398 bp, 223 bp and 125 bp, respectively. We configured three reaction systems with HUV-EC-C cell total DNA as the HHV-6 positive template. The specific amplification reaction system is shown in Table 5.

[0078] The electrophoresis results after reaction are shown in Figure 4As shown in A, the target fragment of HHV-6 in the first experimental group is 398 bp, but the target fragment is not amplified in the system; while the target fragments of HHV-6 in the second and third experimental groups are successfully amplified, which are 223 bp and 125 bp respectively. Considering the size distribution of the target fragments, the primer combination of the second experimental group is selected for subsequent experiments.

[0079] To further verify the specificity of the primers selected by the seven-PCR detection system, we mixed the seven pairs of primers in equal proportions to prepare a primer group, and added single positive templates of seven viruses and mixed positive templates of seven viruses respectively for reaction. The electrophoresis results are as follows Figure 4 As shown in B, it shows that the system can simultaneously detect seven human herpes viruses, and has strong specificity.

[0080] (5) Establishment of eight-PCR system

[0081] On the basis of the above-mentioned seven-PCR reaction system, HHV-7 primers were added to the primer combination, and the corresponding target fragment size was 114 bp. HHV-7 plasmid standard was added as a positive template. The specific amplification reaction system is shown in Table 5.

[0082] Table 5 Five-, six-, seven- and eight-PCR reaction systems

[0083] At this time, the system contains eight pairs of primers and eight virus positive templates, the components are complex, and the band size of HHV-7 and EBV is relatively close. In order to simultaneously amplify 8 target fragments and ensure clear bands, the EBV primers were replaced after repeated attempts, and the size of the amplified target fragment was adjusted to 85 bp. Finally, an eight-PCR detection system was established.

[0084] The results are shown in Figure 5 As shown, the amplified fragments of each virus meet the expected size, and there is no non-specific amplification, indicating that the system can simultaneously detect 8 human herpes viruses, and the effect is good.

[0085] The finally obtained primer combination for the eight-PCR detection system is shown in Table 6.

[0086] Table 6 Eight-PCR detection primer sequence

[0087] Example 3: Preparation of virus standard

[0088] 1. Target fragment amplification

[0089] (1) Design of target fragment amplification primer

[0090] Through literature retrieval and NCBI alignment, based on HSV-1 gene (NC_001806.2), HSV-2 gene (NC_001798.2), VZV gene (NC_001348.1), EBV gene (NC_001847.1), HCMV gene (NC_006273.2), HHV-6 gene (NC_001664.4), HHV-7 gene (NC_001716.2) and HHV-8 gene (NC_009333.1), detection primers were designed. The specific design idea of the primers is: the length of the primers is 25-30 bp, the annealing temperature is 55-65℃, the GC content is 35%-50%, and then the designed primers are compared and analyzed by the Primer Blast module of NCBI. The sequences of the target fragments amplified by the primers of the eight human herpes viruses are shown as SEQ ID NO. 1-8.

[0091] (2) Target fragment amplification and gel recovery

[0092] PCR amplification was performed with the target fragment sequences shown in SEQ ID NO. 1-8 as templates, and the target fragment amplification products were obtained by agarose gel recovery, sequencing, and determination of no error before proceeding to the next step.

[0093] 2, Ligation transformation

[0094] (1) Double enzyme digestion of target fragment and vector

[0095] The target fragment and pMD18-T plasmid were double enzyme-digested, the enzyme digestion products (linearized pMD18-T plasmid vector and target fragment) were purified, and were prepared for use.

[0096] (2) Ligation of target fragment and linearized vector

[0097] The target fragment and linearized vector were ligated to obtain a ligation product (recombinant expression vector containing the target fragment) for use.

[0098] (3) Transformation and screening identification

[0099] The ligation product was transformed into competent cells, and the successfully transformed DH5a was screened and preserved for use.

[0100] 3, Plasmid extraction and standard preparation

[0101] The plasmid of the above successfully transformed DH5a was extracted, sequenced, and identified as correct. The molecular weight and concentration of the plasmid were calculated, and the concentration was gradually diluted from 10 10 copies / μL to 10 1 copies / μL, a total of 10 concentration gradients. 10 μL of plasmid with a concentration of 1×10 10copies / μL of the standard mixture.

[0102] Example 4: Sensitivity determination

[0103] To further explore the sensitivity of the octuple PCR system for simultaneous detection of 8 human herpes viruses, each virus plasmid was diluted, and the sensitivity of single virus and mixed viruses in the octuple PCR system was determined. The eight virus plasmids that had been constructed were gradient diluted with ddH2O to 1 x 10 7 , 1 x 10 6 , 1 x 10 5 , 1 x 10 4 , 1 x 10 3 , 1 x 10 2 , 1 x 10 1 , 1 x 10 0 copies / μL. In the single virus sensitivity determination, 1 μL of the corresponding virus plasmid was taken from each virus concentration gradient as a template and added to the mixed primer system for amplification. In the sensitivity determination of mixed viruses, 1 μL of each of the 8 plasmids in each gradient was taken as a template for amplification, and 10 μL of the product was taken after the reaction for agarose gel electrophoresis (130 V, 220 A) to observe the results.

[0104] The results of agarose gel electrophoresis are shown in Figure 6 When the plasmid was diluted to ≥ 1 x 10 4 copies, a clear band could be observed; but when diluted to ≤ 1 x 10 3 copies, no clear band could be observed. Therefore, the lower limit of the sensitivity of the octuple PCR method for simultaneous detection of 8 herpes viruses is 1 x 10 4 copies (50 μL system).

[0105] Under the premise that the octuple PCR detection system established in the present application can simultaneously detect 8 human herpes viruses, considering that the cultured cells may be infected with only one or a few viruses, the sensitivity of the octuple PCR detection system for detecting any one herpes virus was also studied. Specifically, 1 μL of each of the 8 positive plasmids was taken as a template and added individually to the mixed primer system for amplification.

[0106] The results of agarose gel electrophoresis are shown in Figure 7 It can be seen that for HSV-1, VZV, HCMV, HHV-6, HHV-8, HSV-2 and EBV, when the plasmid copy number is ≥ 1 x 10 4 , these 7 viruses can be detected individually, but when diluted to 1 x 10 3or lower, no obvious band can be observed by electrophoresis; for HHV-7, when the plasmid is diluted to 1×10 3 copies / μL, the electrophoresis result can still observe a band. Therefore, the lower limit of the eight-plex PCR method constructed in the present application for detecting the copy number of HHV-7 virus is 1×10 3 copies / μL, and the lower limit of the copy number of the remaining seven viruses is 1×10 4 copies / μL.

[0107] Example 5: Specificity verification

[0108] Using B95-8 (EBV), BCBL-1 (HHV-8) and HUV-EC-C (HHV-6) cell DNA as a single template, respectively, and adding single detection primer pairs and mixed primer combinations of eight viruses, amplification was carried out at 59°C; after the reaction was completed, 10 μL of the mixture was taken for agarose gel electrophoresis (130 V, 220 A), and the amplification results of the single primer and the mixed primer combination were observed and compared.

[0109] The results are shown in Figure 8 . Taking the experiment with B95-8 cell DNA as the template as an example, as shown in Figure 8 A, only when the EBV primer is added, the target band can be detected, and the rest are negative. The same is true for the other two groups of experiments, proving that the single primer and the mixed primer combination are specific, and further verifying the specificity of the eight-plex PCR system.

[0110] Example 6: Optimization of reaction conditions of eight-plex PCR system

[0111] 1. Optimization of annealing temperature

[0112] The reaction program of the eight-plex PCR system is: (1) 95°C pre-denaturation for 1 min; (2) 95°C denaturation for 23 min, 55-63°C annealing for 30 s, 72°C extension for 1 min; a total of 35 cycles; (3) 72°C thorough extension for 10 min, 4°C storage for standby. Among them, the annealing temperature is crucial to the PCR amplification effect. In order to explore the optimal annealing temperature of the system, in the optimization experiment of the annealing temperature, the positive templates of the viruses are all 1×10 5 copies / μL of plasmid standard. According to the annealing temperature of the selected eight pairs of primers, five annealing temperature gradients are set: 55°C, 57°C, 59°C, 61°C and 63°C, and the specific binding of the primers to the template and the amplification efficiency under different annealing temperatures are observed.

[0113] The results of gel electrophoresis are shown in Figure 9As shown in the figure, VZV, HHV-8, HSV-2, HHV-7, and EBV showed stable amplification across the tested temperature range. However, the target bands for HSV-1 and HHV-6 gradually weakened with increasing temperature, while the target band for HCMV gradually became clearer. Considering the amplification performance of all eight fragments, 59°C was ultimately selected as the optimal annealing temperature.

[0114] 2. Optimization of primer concentration

[0115] Due to the complex components of the octaplex PCR system, we continuously adjusted and optimized the primer concentrations of each pair to ensure uniform band clarity and amplification efficiency for all eight viral targets. Based on previous experimental results, the primer concentrations of the eight primer pairs were adjusted for each reaction. 10 μL of the product was analyzed by agarose gel electrophoresis (130 V, 220 A) to observe the electrophoresis results.

[0116] The results are as follows Figure 10 As shown in the figure, the brightness of the amplified specific bands remains consistent, whether the system detects only one herpes virus or all eight simultaneously. Therefore, in the octal PCR system, the primer concentrations for VZV are 150 nM, those for HSV-1 and EBV are 300 nM, and the primer concentrations for the remaining five viruses are all 200 nM.

[0117] Comparative Example 1: Construction of an eight-layer RPA reaction system

[0118] The system uses the primers of the octaplex PCR system and prepares them into 10 μM, 15 μM, 20 μM, 25 μM and 30 μM working solutions respectively, and then mixes them in equal volumes, and takes a concentration of 1×10 5 Eight plasmids were mixed at equal volumes, with a concentration of 8 copies / μL. Five octaplex RPA reaction systems were configured according to the TwistAmp® Basic kit instructions.

[0119] To the reaction tube, add 29.5 μL of buffer, 4 μL of primer mix, 4 μL of template, and 10 μL of ddH2O. Finally, add 2.5 μL of activator to initiate the reaction. Mix thoroughly and incubate in a 39°C metal bath for 20 minutes. After amplification, remove a portion of the sample for purification. Then, add 5 μL of 10× loading buffer and mix thoroughly. Load 10 μL of the sample onto a well of a 3% agarose gel and perform electrophoresis at 120 V, 200 A to observe and compare the amplification of the target band.

[0120] The results are as follows Figure 11 As shown in Figure A, only three relatively obvious fragments were observed. The amplified product was purified and subjected to gel electrophoresis. The results are as follows Figure 11As shown in Figure B, many non-specific amplification bands appeared. In addition, we redesigned the RPA primers and tried a lot of times, but the octuple RPA detection system was not successfully constructed.

[0121] Application Example: Application of the Octuple PCR Detection System in Cells

[0122] In this application example, the octuple PCR detection system described above was used to detect human herpes viruses in some cells preserved by the China Center for Type Culture Collection (CCTCC).

[0123] A total of 30 cells were detected in this experiment, covering various cell types and sources. The agarose gel electrophoresis results are shown in Figure 1. Figure 12 As can be seen, among the 30 cells, the ARH-77 cell showed a specific band of EBV positive, indicating that the cell was infected with EBV, and the remaining 29 cells did not detect any specific band of herpes virus, indicating that these cells were negative for herpes virus.

[0124] The above specific embodiments describe the implementation of the present application, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A primer combination for simultaneous detection of eight human herpes viruses in a single tube, characterized in that: The primer combination consists of a forward primer and a reverse primer of the nucleotide sequence shown in SEQ ID NO.9 to SEQ ID NO.24, and the primer combination is used to simultaneously detect human herpes viruses HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7 and HHV-8 in the same tube.

2. An octal PCR detection system for simultaneous detection of eight human herpes viruses in a single tube, characterized in that: Comprising the primer combination according to claim 1.

3. The single-tube octal PCR detection system for simultaneous detection of eight human herpes viruses according to claim 2, characterized in that: Also included are plasmid standards for detecting eight viruses: HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, and HHV-8.

4. The single-tube octal PCR detection system for simultaneous detection of eight human herpes viruses according to claim 3, characterized in that: The plasmid standards of 8 viruses contain the target fragments of 8 viruses: HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7 and HHV-8.

5. A product for simultaneous detection of eight human herpes viruses in a single tube, characterized in that: The method comprises the primer combination according to claim 1, or the octaplex PCR detection system according to any one of claims 1 to 4.

6. A method for simultaneously detecting eight human herpes viruses in a single tube, not for the purpose of disease diagnosis and treatment, characterized in that: The primer combination of claim 1, or the eight-fold PCR detection system of any one of claims 1 to 4, or the product of claim 5 is used to simultaneously detect eight human herpes viruses in a sample to be tested in a single tube.

7. The method for simultaneously detecting eight human herpes viruses in a single tube without the purpose of disease diagnosis and treatment according to claim 6, characterized in that: In the primer combination, the concentration of the forward primer and reverse primer for detecting VZV virus is 150 nM, the concentration of the forward primer and reverse primer for detecting HSV-1 and EBV virus is 300 nM, and the concentration of the forward primer and reverse primer for detecting HSV-2, HCMV, HHV-6, HHV-7 and HHV-8 virus is 200 nM.

8. The method for simultaneously detecting eight human herpes viruses in a single tube without the purpose of disease diagnosis and treatment according to claim 6, characterized in that: When the primer combination is used for PCR amplification, the annealing temperature is 55-63°C; Furthermore, the annealing temperature is 59°C.

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