CN112442553A - Kit and method for JCPyV detection and typing - Google Patents

Kit and method for JCPyV detection and typing Download PDF

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CN112442553A
CN112442553A CN202011164715.2A CN202011164715A CN112442553A CN 112442553 A CN112442553 A CN 112442553A CN 202011164715 A CN202011164715 A CN 202011164715A CN 112442553 A CN112442553 A CN 112442553A
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jcpyv
typing
detection
kit
probe
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Inventor
欧铜
徐银燕
吴松
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Shenzhen Luohu Peoplel's Hospital
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention discloses a kit and a method for JCPyV detection and typing, wherein the kit comprises an upstream primer with a sequence shown in SEQ ID.1, a downstream primer with a sequence shown in SEQ ID.2, a first probe with a sequence shown in SEQ ID.3 and a second probe with a sequence shown in SEQ ID.4, wherein the first probe is used for being combined with a JCPyV wild type NCCR sequence, and the second probe is used for being combined with a JCPyV mutant type NCCR sequence. The kit provided by the invention can effectively detect JCPyV and divide the JCPyV into wild type and mutant type, has the advantages of simple operation, high sensitivity, high specificity, low cost and the like, and is very suitable for rapid detection and parting of clinical JCPyV.

Description

Kit and method for JCPyV detection and typing
Technical Field
The invention relates to the field of virus detection, in particular to a kit and a method for JCPyV detection and typing.
Background
JCPyV belongs to polyomaviridae, and has a circular double-stranded DNA genome with a total length of about 5.1 kb. The genome can be roughly divided into an early coding region, a late coding region and a non-coding gene control region (NCCR) according to the action of the gene sequence and the expression timing of the encoded product. NCCR is a variable region of the JCPyV genome, approximately 400bp in size, and includes viral DNA replication initiation sites, viral protein transcription TATA boxes, host cell DNA and transcription factor binding sites, promoter and enhancer elements, and viral large T antigen binding sites.
JCPyV undergoes virulent infection in some cells (such as glial cells) expressing its replicating transcription factor, replicates the viral genome, transcribes and expresses viral structural proteins, assembles progeny viruses, lyses host cells, and causes disease in cases of severe immune impairment in the body, such as progressive multifocal leukoencephalopathy. In most cases, JCPyV is invisible infection, no obvious clinical symptoms exist, immune evaded virus particles are usually hidden in epithelial tissues of the urinary system, and virus nucleic acid can be detected in urine of partial healthy adults. At present, JCPyV genome and large T antigen expression are detected in various tumor tissues such as brain cancer, colorectal cancer, gastric cancer and the like. Epidemiological studies and Mata analysis show that chronic infection of pathogenic microorganisms such as viruses is one of the important risk factors for the development of bladder cancer. At present, the rapid detection of JCPyV in urine and the typing of bladder cancer specific NCCR mainly depend on PCR amplification and Sanger sequencing, so that the rapid detection and analysis in a medical laboratory are inconvenient.
Therefore, the development of a rapid detection kit for rapid detection of JCPyV and non-coding region typing has important application value.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a kit and a method for JCPyV detection and typing, which are simple in operation, high in sensitivity, high in specificity and low in cost.
The technical scheme of the invention is as follows:
a kit for JCPyV detection and typing comprises an upstream primer with a sequence shown as SEQ ID.1, a downstream primer with a sequence shown as SEQ ID.2, a first probe with a sequence shown as SEQ ID.3 and a second probe with a sequence shown as SEQ ID.4, wherein the first probe is used for being combined with a JCPyV wild-type NCCR sequence, and the second probe is used for being combined with a JCPyV mutant-type NCCR sequence.
The kit for JCPyV detection and typing is characterized in that the 5 'end of the first probe is connected with a fluorescence reporter group HEX, and the 3' end of the first probe is connected with a fluorescence quenching group BHQ-X.
The kit for JCPyV detection and typing is characterized in that the 5 'end of the second probe is connected with a fluorescence reporter group FAM, and the 3' end of the second probe is connected with a fluorescence quenching group BHQ-X.
The kit for JCPyV detection and typing further comprises 10-star PCR buffer solution and Mg2+dNTPs and HSTaq enzyme.
A method for JCPyV detection and typing, comprising the steps of:
carrying out PCR amplification on a sample to be detected by using the kit disclosed by the invention to obtain an amplified sample;
in the PCR amplification process, an SLAN full-automatic medical PCR analysis system is adopted to automatically acquire data to simulate and generate an amplification curve;
and detecting and typing JCPyV in the sample to be detected according to the generated amplification curve.
Has the advantages that: the invention provides a kit for JCPyV detection and typing, which can effectively detect JCPyV and classify the JCPyV into a wild type and a mutant type, has the advantages of simple operation, high sensitivity, high specificity, low cost and the like, and is very suitable for detection and typing diagnosis of the JCPyV in clinic.
Drawings
FIG. 1 is a flow chart of a preferred embodiment of a JCPyV detection and typing method of the present invention.
FIG. 2 is a standard curve plotted according to the JCPyV wild-type assay in example 2 of the present invention.
FIG. 3 is a standard curve chart plotted according to the JCPyV mutant type detection result in example 2 of the present invention.
FIG. 4 is a graph showing the amplification of the JCPyV wild-type in example 3 of the present invention.
FIG. 5 is a graph showing the amplification of JCPyV mutants in example 3 of the present invention.
FIG. 6 is a diagram showing the results of agarose gel electrophoresis detection of PCR products of 16 samples in example 4 of the present invention.
FIG. 7 is a graph showing the fluorescence amplification curve of non-bladder cancer sample-1 in example 4 of the present invention.
FIG. 8 is a graph showing the results of sequencing and typing of non-bladder cancer sample-1 in example 4 of the present invention.
FIG. 9 is a graph showing the fluorescence amplification curve of bladder cancer sample-2 in example 4 of the present invention.
FIG. 10 is a graph showing the results of sequencing and typing of bladder cancer sample-2 in example 4 of the present invention.
Detailed Description
The invention provides a kit and a method for JCPyV detection and typing, and the invention is further detailed below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Based on the characteristics of the NCCR sequence, JCPyV can be typed. The JCPyV infection condition in urine of physical examination healthy people and bladder cancer patients is detected, and the existence of tumor-specific mutation in JCPyV NCCR in urine of bladder cancer patients can be revealed. In the invention, the JCPyV NCCR sequence universally existing in the urine of healthy people is an NCCR wild type, and the JCPyV NCCR sequence detected in the urine of bladder cancer patients is an NCCR mutant type; the sequences of the NCCR wild type and mutant are presented in the summary of the invention. The NCCR sequence variation has been reported to influence the combination with cell transcription regulating factor and has great significance in the development of tumor. At present, the rapid detection of JCPyV in urine and the typing of bladder cancer specific NCCR mainly depend on PCR amplification and Sanger sequencing, so that the rapid detection and analysis in a medical laboratory are inconvenient. Therefore, the development of a rapid detection method and a kit for rapid detection of JCPyV and non-coding region typing has important application value.
Based on the JCPyV detection and typing kit, the JCPyV detection and typing kit comprises an upstream primer with a sequence shown in SEQ ID.1, a downstream primer with a sequence shown in SEQ ID.2, a first probe with a sequence shown in SEQ ID.3 and a second probe with a sequence shown in SEQ ID.4, wherein the first probe is used for being combined with a JCPyV wild type NCCR sequence, and the second probe is used for being combined with a JCPyV mutant type NCCR sequence.
In this embodiment, the sequence of the upstream primer is shown in SEQ id.1, and specifically: 5 'TGGCCTCCTAAAAAGCCTCC 3'; the sequence of the downstream primer is shown as SEQ ID.2, and specifically comprises the following components: 5 'GGTGACAAGCCAAAACAGCTC 3'; the sequence of the first probe is shown as SEQ ID.3, and specifically comprises the following steps: 5 'HEX-AACATGTTCCCCTGGCT-BHQ-X3'; the sequence of the second probe is shown as SEQ ID.4, and specifically comprises the following steps: 5 'FAM-CAAAACATGCTGGCTG-BHQ-X3'.
In this example, the first probe (which may be named JCVWP) was used to bind to a JCPyV wild-type NCCR sequence as shown in SEQ id.5, specifically: TTTTGCTTTTTGTAGCAAAAAATTAGTGCAAAAAAGGGAAAAACAAGGGAATTTCCCTGGCCTCCTAAAAAGCCTCCACGCCCTTACTACTTCTGAGTAAGCTTGGAGGCGGAGGCGGCCTCGGCCTCCTGTATATATAAAAAAAAGGGAAGGTAGGGAGGAGCTGGCTAAAACTGGATGGCTGCCAGCCAAGCATGAGCTCATACCTAGGGAGCCAACCAGCTGACAGCCAGAGGGAGCCCTGGCTGCATGCCACTGGCAGTTATAGTGAAACCCCTCCCATAGTCCTTAATCACAAGTAAACAAAGCACAAGGGGAAGTGGAAAGCAGCCAGGGGAACATGTTTTGCGAGCCAGAGCTGTTTTGGCTTGTCACCAGCTGGCC are provided. The second probe (named JCVMP) is used for combining a JCPyV mutant NCCR sequence, and the JCPyV mutant NCCR sequence is shown as SEQ ID.6 and specifically comprises the following components: TTTTGCTTTTTGTAGCAAAAAATTAGTGCAAAAAAGGGAAAAACAAGGGAATTTCCCTGGCCTCCTAAAAAGCCTCCACGCCCTTACTACTTCTGAGTAAGCTTGGAGGCGGAGGCGGCCTCGGCCTCCTGTATATATAAAAAAAAGGGAAGGTAGGGAGGAGCTGGCTAAAACTGGATGGCTGCCAGCCAAGCATGAGCTCATACCTAGGGAGCCAACCAGCTGACAGCCAGAGGGAGCCCTGGCTGCATGCCACTGGCAGTTATAGTGAAACCCCTCCCATAGTCCTTAATCACAAGTAAACAAAGCACAAGGGGAAGTGGAAAGCAGCCAGCATGTTTTGCGAGCCAGAGCTGTTTTGGCTTGTCACCAGCTGGCC are provided.
In this embodiment, the 5 'end of the first probe is connected with a fluorescence reporter group HEX, and the 3' end of the first probe is connected with a fluorescence quenching group BHQ-X; the 5 'end of the second probe is connected with a fluorescence reporter group FAM, and the 3' end of the second probe is connected with a fluorescence quenching group BHQ-X.
In some embodiments, the kit further comprises 10 × PCR buffer, Mg2+dNTPs and HSTaq enzyme.
In some specific embodiments, the kit for JCPyV detection and typing comprises a reaction system as shown in table 1:
TABLE 1 reaction System
Figure BDA0002745410510000051
Figure BDA0002745410510000061
In some embodiments, based on the kit, there is also provided a method for JCPyV detection and typing, as shown in fig. 1, comprising the steps of:
s10, carrying out PCR amplification on a sample to be detected by adopting the kit disclosed by the invention to obtain an amplified sample;
s20, automatically collecting data by using an SLAN full-automatic medical PCR analysis system in the PCR amplification process to generate an amplification curve in a simulation way;
and S30, typing the JCPyV in the sample to be detected according to the generated amplification curve.
In this example, the reaction procedure for the PCR amplification is as follows: 1. reacting for 3min at 94 ℃; 2. 40 cycles were performed, each cycle comprising: the reaction was carried out at 94 ℃ for 20sec and at 60 ℃ for 30 sec.
The kit and method for JCPyV detection and typing of the present invention are further explained by the following specific examples:
example 1
Providing a kit for JCPyV detection and typing, which comprises an upstream primer, a downstream primer, JCVWP and JCVMP; the 5 'ends of the JCVWP and the JCVMP are respectively connected with a fluorescence reporter group HEX and a FAM, and the 3' ends of the JCVWP and the JCVMP are connected with a fluorescence quenching group BHQ-X.
An upstream primer: 5 'TGGCCTCCTAAAAAGCCTCC 3'
A downstream primer: 5 'GGTGACAAGCCAAAACAGCTC 3'
JCVWP：5’HEX-AACATGTTCCCCTGGCT-BHQ-X 3’
JCVMP：5’FAM-CAAAACATGCTGGCTG-BHQ-X 3’。
The primers and probes were synthesized by bio-corporation and configured as described.
Example 2
JCPyV infection and typing was detected using the kit described in example 1
1. Reaction system configuration
The reaction system was prepared as shown in Table 1, and was prepared on ice under strict dark conditions, followed by the above-mentioned reaction procedure. The DNA template is a sample obtained by diluting JCPyV wild type plasmids and mutant plasmids according to copy numbers of 105copy/ul, 104copy/ul, 103copy/ul, 102copy/ul and 10copy/ul respectively.
2. Criteria for judging results
The kit can be used for detecting the wild type and the mutant type of the NCCR sequence of the JCPyV, thereby diagnosing the JCPyV infection condition. The judgment standard of the detection result is as follows:
(1) JCPyV wild type: the amplification curve of JCVWP is smooth and is in an S shape; CT value is less than 40;
(2) JCPyV mutant: the amplification curve of the JCVMP is smooth and is in an S shape; the CT value is less than 40.
3. The result of the detection
In the plasmids of the JCPyV wild type and the JCPyV mutant type, the plasmids with copy numbers of all concentrations have a smooth amplification curve and are in an S shape; and the CT values are all less than 40, and the detection results all accord with the judgment standard. The results of the detection of the JCPyV wild type and mutant type are plotted as standard curves, respectively, and are shown in FIGS. 2 and 3.
Example 3
Investigating the detection limit of sequence copy number in a reaction System
And (3) carrying out repeated detection on the JCPyV wild type plasmid and the JCPyV mutant plasmid diluted into concentration gradient samples of 10copy/ul, 20copy/ul and 40copy/ul respectively, wherein the corresponding concentration gradient is the detection limit when at most 1 hole in 20 repeated holes cannot be amplified. The amplification curve of the JCPyV wild type and the amplification curve of the JCPyV mutant type in the sample with the plasmid concentration of 20copy/ul are shown in FIGS. 4 and 5, respectively. The results show that the detection limit of the wild type and the mutant type are both 20 copy/ul.
Example 4
In this example, JCPyV was detected and typed in 16 clinical specimens (8 patients with bladder cancer and 8 patients with non-bladder cancer). After extracting DNA from the urine of the subject, the kit of example 1 was used to perform the detection method described in table 1, and the PCR products of 16 samples were subjected to agarose gel electrophoresis (as shown in fig. 6) and first-generation sequencing. The results of the assay showed that 7 of the 16 samples had JCPyV infection, and the typing results are shown in Table 2. Sequencing results show that the sequence has obvious set peaks after the deletion mutation of the NCCR sequence.
TABLE 2 test results
Figure BDA0002745410510000081
Figure BDA0002745410510000091
In the Table 2, the fluorescence amplification curve of the non-bladder cancer sample-1 is shown in FIG. 7, and the sequencing typing result of the non-bladder cancer sample-1 is shown in FIG. 8; the fluorescence amplification curve of the bladder cancer sample-2 is shown in FIG. 9, and the sequencing typing result of the bladder cancer sample-2 is shown in FIG. 10. As can be seen from FIG. 7, only one fluorescence amplification sigmoid curve was observed, the CT value was 24.13 and was less than 40, and the amplification sigmoid curve was an amplification curve of JCVWP (HEX fluorescence) as seen in the software program, so that it was determined that the NCCR sequence containing JCPyV in the non-bladder cancer sample-1 was wild-type. From the sequencing results in FIG. 8, it was found that the NCCR sequence containing JCPyV in the non-bladder cancer sample-1 was wild-type. The kit provided by the invention is proved to be suitable for rapid detection and typing of clinical JCPyV.
As can be seen from fig. 9, there are two fluorescence amplification sigmoid curves, both of which have CT values less than 40, and the two sigmoid curves are respectively the amplification curve of JCVWP (HEX fluorescence) and the amplification curve of JCVMP (FAM fluorescence) in the software program, so that it can be determined that the non-bladder cancer sample-2 contains both the wild-type JCPyV NCCR sequence and the mutant-type JCPyV NCCR sequence. From the sequencing results in FIG. 10, it was also found that the non-bladder cancer sample-2 contained both the wild-type JCPyV NCCR sequence and the mutant-type JCPyV NCCR sequence. The kit provided by the invention is proved to be suitable for rapid detection and typing of clinical JCPyV.
In conclusion, the invention provides a kit for JCPyV detection and typing, the kit can effectively detect JCPyV and classify the JCPyV into a wild type and a mutant type, has the advantages of simple operation, high sensitivity, high specificity, low cost and the like, and is suitable for rapid detection and typing of clinical JCPyV.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
<110> Shenzhen Luhu regional people hospital
<120> kit and method for JCPyV detection and typing
<160> 6
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (rengongxulie)
<400> 1
tggcctccta aaaagcctcc 20
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (rengongxulie)
<400> 2
ggtgacaagc caaaacagct c 21
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (rengongxulie)
<400> 3
aacatgttcc cctggct 17
<210> 4
<211> 26
<212> DNA
<213> Artificial sequence (rengongxulie)
<400> 4
caaaacatgc tggctg 16
<210> 5
<211> 334
<212> DNA
<213> Artificial sequence (rengongxulie)
<400> 5
ttttgctttt tgtagcaaaa aattagtgca aaaaagggaa aaacaaggga atttccctgg cctcctaaaa 60
agcctccacg cccttactac ttctgagtaa gcttggaggc ggaggcggcc tcggcctcct gtatatataa 120
aaaaaaggga aggtagggag gagctggcta aaactggatg gctgccagcc aagcatgagc tcatacctag 180
ggagccaacc agctgacagc cagagggagc cctggctgca tgccactggc agttatagtg aaacccctcc 240
catagtcctt aatcacaagt aaacaaagca caaggggaag tggaaagcag ccaggggaac atgttttgcg 300
agccagagct gttttggctt gtcaccagct ggcc 334
<210> 6
<211> 329
<212> DNA
<213> Artificial sequence (rengongxulie)
<400> 6
ttttgctttt tgtagcaaaa aattagtgca aaaaagggaa aaacaaggga atttccctgg cctcctaaaa 60
agcctccacg cccttactac ttctgagtaa gcttggaggc ggaggcggcc tcggcctcct gtatatataa 120
aaaaaaggga aggtagggag gagctggcta aaactggatg gctgccagcc aagcatgagc tcatacctag 180
ggagccaacc agctgacagc cagagggagc cctggctgca tgccactggc agttatagtg aaacccctcc 240
catagtcctt aatcacaagt aaacaaagca caaggggaag tggaaagcag ccagcatgtt ttgcgagcca 300
gagctgtttt ggcttgtcac cagctggcc 329

Claims (5)

1. A kit for JCPyV detection and typing is characterized by comprising an upstream primer with a sequence shown as SEQ ID.1, a downstream primer with a sequence shown as SEQ ID.2, a first probe with a sequence shown as SEQ ID.3 and a second probe with a sequence shown as SEQ ID.4, wherein the first probe is used for being combined with a JCPyV wild type NCCR sequence, and the second probe is used for being combined with a JCPyV mutant type NCCR sequence.
2. The kit for JCPyV detection and typing according to claim 1, wherein the first probe has a fluorescent reporter group HEX connected to the 5 'end and a fluorescent quencher group BHQ-X connected to the 3' end.
3. The kit for JCPyV detection and typing according to claim 1, wherein the second probe has a fluorescent reporter group FAM connected to the 5 'end and a fluorescence quencher group BHQ-X connected to the 3' end.
4. The kit for JCPyV detection and typing according to claim 1, further comprising 10-PCR buffer, Mg2+dNTPs and HSTaq enzyme.
5. A method for JCPyV detection and typing comprising the steps of:
carrying out PCR amplification on a sample to be detected by using the kit disclosed by the invention to obtain an amplified sample;
in the PCR amplification process, an SLAN full-automatic medical PCR analysis system is adopted to automatically acquire data to simulate and generate an amplification curve;
and detecting and typing JCPyV in the sample to be detected according to the generated amplification curve.
CN202011164715.2A 2020-10-27 2020-10-27 Kit and method for JCPyV detection and typing Pending CN112442553A (en)

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