CN113462685B - Probe composition for preventing reverse transcription of fungus conserved region and application thereof - Google Patents
Probe composition for preventing reverse transcription of fungus conserved region and application thereof Download PDFInfo
- Publication number
- CN113462685B CN113462685B CN202110827350.5A CN202110827350A CN113462685B CN 113462685 B CN113462685 B CN 113462685B CN 202110827350 A CN202110827350 A CN 202110827350A CN 113462685 B CN113462685 B CN 113462685B
- Authority
- CN
- China
- Prior art keywords
- artificial sequence
- dna
- rrna
- rna
- reverse transcription
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a probe composition for preventing reverse transcription of fungus conserved regions, which comprises 114 probes aiming at 18S rRNA and 25S rRNA sequence conserved regions, wherein the sequences of the 114 probes are shown as SEQ ID NO.1-114, each probe can be combined with not less than 10% of common fungus rRNA, can specifically and efficiently identify and combine the conserved sequence regions of the target fungus rRNA, prevents reverse transcription of RNA of the regions, and simultaneously retains normal reverse transcription of rRNA of non-conserved regions. The fungus rRNA reverse transcription probe disclosed by the invention has the advantages of simplicity in operation (one-step operation), short time consumption (2 min), small loss, low cost and the like, remarkably improves the effective data duty ratio, detection rate, sensitivity and accuracy of the RNA mNSS detection technology, and is very suitable for the automatic detection of the RNA mNSS.
Description
Technical Field
The invention relates to a probe composition for preventing reverse transcription of a fungus conserved region and application thereof in RNA (ribonucleic acid) database establishment, belonging to the technical field of biology.
Background
Pathogenic microorganism detection is extremely important in diagnosis of infectious diseases, and traditional detection methods mainly comprise separation culture and biochemical identification, smear microscopy, immunological methods, PCR detection and gene chip technology, which can only detect known pathogenic microorganisms, but cannot effectively diagnose unknown symptoms. With the rise in the field of intestinal microorganisms and pathogenic microorganisms, many diseases or conditions are the result of the co-action of a variety of microorganisms. Therefore, high-throughput genomics technology and high-throughput transcriptomics technology are used to systematically identify microbial species in pathological samples, providing a powerful diagnostic tool for the diagnosis of disease caused by pathogenic microorganisms, called metagenomic new generation sequencing technology (metagenomics next generation sequencing, mNGS). The technology is characterized in that DNA/RNA in clinical samples is directly extracted without culture and preference, and detection of pathogens such as bacteria, fungi, viruses and parasites is completed at one time by adopting a high-throughput sequencing technology and through database comparison and biological analysis. However, the large proportion of fungal ribosomal RNA in pathogenic microorganisms and the high conservation of these rRNAs in fungi make it difficult to distinguish which pathogenic bacteria are the source of these same regions and occupy the sequencing data, resulting in less useful data. This not only increases the amount of sequencing data and the sequencing cost, but also reduces the efficiency and accuracy of pathogenic microorganism detection.
Disclosure of Invention
The object of the present invention is to provide a probe composition for preventing reverse transcription of fungal conserved regions, each probe being capable of binding not less than 10% of common fungal rRNAs, capable of specifically and efficiently recognizing and binding to conserved sequence regions of these target fungal rRNAs, and preventing reverse transcription of RNAs of these regions, while retaining normal reverse transcription of rRNAs of non-conserved regions.
The invention adopts the technical scheme that: a probe composition for reverse transcription of fungus 18S rRNA conserved region is characterized by being a probe mixture shown in the following table
Preferably, the base of the italicized underlined part in the probe sequence is LNA modified base, 3' -terminal-NH 2 C 6 And (5) sealing.
The invention also discloses a probe composition for reverse transcription of fungus 25S rRNA conserved region, which is characterized by being a probe mixture shown in the following table
Preferably, the base of the italicized underlined part in the probe sequence is LNA modified base, 3' -terminal-NH 2 C 6 And (5) sealing.
The invention also discloses application of the probe composition in RNA library construction.
The action mechanism of the invention: contacting the sample total RNA molecules with the probe composition and random primers in a reverse transcription buffer under high temperature conditions such that the probes form hybridized duplex with the rRNA molecules, preferably RNA: DNA, therein to obtain a high temperature hybridization mixture.
Cooling the obtained high-temperature hybridization mixture, and contacting the total RNA molecules with the probe composition and the random primers under the low-temperature condition so that the random primers and other RNA molecules form RNA-DNA hybridization double chains to obtain a low-temperature hybridization mixture; the Tm value and the binding stability of the probe composition after modification by locked nucleic acid are far higher than those of random primers, so that the probe composition can be combined with rRNA under high temperature conditions. While the random primer has a very low Tm, it can bind to other RNAs only at low temperature.
The hybridization mixture at low temperature generated in the above steps is contacted with reverse transcriptase to generate a strand cDNA. rRNA cannot be removed by extension of reverse transcriptase as a template during the generation of a strand of cDNA;
the construction of the downstream RNA NGS library is accomplished by conventional means.
The 18S rRNA and 25S rRNA sequence conservation regions of 246 common fungi recorded in the SILVA database are designed and comprise 114 probes (SEQ ID No. 1-114), each probe can be combined with not less than 10% of common fungus rRNA, can specifically and efficiently identify and combine with the conservation sequence regions of the rRNA of the target fungi, and prevent reverse transcription of RNA in the regions, and meanwhile, normal reverse transcription of rRNA in non-conservation regions is reserved. The fungus rRNA reverse transcription probe disclosed by the invention has the advantages of simplicity in operation (one-step operation), short time consumption (2 min), small loss, low cost and the like, remarkably improves the effective data duty ratio, detection rate, sensitivity and accuracy of the RNA mNSS detection technology, and is very suitable for the automatic detection of the RNA mNSS.
The beneficial effects of the invention are as follows:
1. the invention designs a specific fungal rRNA conserved region reverse transcription blocking probe, which can effectively remove the sequence of rRNA of most overlapped parts in fungi in the reverse transcription process, improves the duty ratio of effective data in the RNA mNGS detection process, reduces the sequencing cost and improves the detection rate of pathogenic microorganisms.
2. The reverse transcription inhibition probe method for removing the fungal rRNA conserved region provided by the invention is simple in operation (one step), short in time consumption (2 min), and very suitable for large-scale industrial automation.
3. The reverse transcription blocking probe method for removing the fungal rRNA conserved region has the advantages of high efficiency, high speed, simple operation, small loss and improvement of the detection rate, sensitivity and accuracy of pathogenic microorganisms.
Drawings
FIG. 1 principle of action of fungal rRNA conserved region reverse transcription blocking probe.
FIG. 2 the ratio of each RNA in the RNA standard sequencing data under three conditions.
FIG. 3 comparison of the detection numbers of fungi at different sequencing depths for RNA standards.
FIG. 4 comparison of fungal detection rates under three conditions.
FIG. 5 comparison of the detection numbers of fungi at different amounts of RNA standard.
FIG. 6 comparison of fungal detection numbers of pathogenic sample RNA under three conditions.
FIG. 7 comparison of fungal detection numbers of different pathogenic sample RNA inputs.
Detailed Description
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification taken in conjunction with the drawings. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever.
EXAMPLE 1 design and preparation of fungal rRNA reverse transcription Barrier removal probes
The sequences and modifications of the probes and primers used in this example are shown in Table 1.
TABLE 1
The probe and primer sequence transcription inhibition removal probe composition can be applied to various high-throughput sequencing platforms, such as Illumina, huada MGI-seq, nanopore or Pacbio, and the like, is applied to detection ranges of transcriptomics, apparent transcriptomics field researches and the like, is used for effectively removing rRNA in pathogenic microorganism sample RNA, and improves the detection rate of pathogenic microorganisms.
The probe composition includes a combination of one or more of the single-stranded DNA probes designed for the 246 fungal rRNA molecule conserved regions recorded in the SILVA database, the probe sequences and modifications are shown in table 1, and the fungal rRNA includes a combination of one or more of 18S rRNA and 25S rRNA.
The length of each single-stranded DNA probe is 20nt-25nt;
the 3' -end of each probe is NH-substituted 2 C 6 Modification and closure, wherein the middle of the modified probe contains a plurality of locked nucleic acid modification sites, and 50% of the locked nucleic acid sites are positioned in the front third area of the 5' end of the probe;
each probe has its own complement value of less than 5 and Tm value of greater than 80 ℃.
According to the above conditions, the fungal rRNA probe composition was designed to contain 114 probes, and the probe sequences and modifications are shown in Table 1.
Table 1 all probes were dissolved in DEPC water to a final concentration of 20. Mu.M, mixed in equal volumes and the concentration of each single stranded DNA probe in the mixture was 0.5-2. Mu.M.
Example 2: and (3) preparing and detecting a human pathogenic RNA standard containing fungal RNA.
In this example, we prepared a human pathogenic RNA standard containing fungal RNA and verified the content ratio of fungi in the standard and the effect of reverse transcription of the fungal rRNA conserved region blocking probe removal of the rRNA conserved region using RNA pool-making sequencing. The specific implementation mode is as follows:
1) Preparation of RNA standard mixture: using assist organismsCell/Tissue Total RNA Kit (Cat# 19211) extracts RNA from 293F cells. RNA from Pichia pastoris and Streptomyces roseoflorius was extracted using the merck fungal purification kit (Cat#E4913-1 KT). Other 10 kindsFungal RNA is derived from the genetic technology of Mallotus philippinensis, including bran Ma Laxian, physcomitrella, cladosporium californicum, pei Shi, trichosporon verrucosum, trichosporon rosenbergii, cryptococcus neoformans, aspergillus flavus, mucor, and Pneumocystis.
Standard concentrations were determined using Nanodrop. The above RNAs were mixed in the proportions shown in the following table:
TABLE 2
| Component (A) | Dosage of |
| Human 293F RNA | 100μg |
| Pichia pastoris RNA | 1000ng |
| Mucor RNA | 100ng |
| Streptomyces roseoflorius RNA | 10ng |
| Aspergillus flavus RNA | 1ng |
| Physcomitrella mentagrophytes RNA | 0.1ng |
| Mallotus furfur Ma Laxian bacterial RNA | 0.01ng |
| Cladosporium californicumRNA | 0.01ng |
| Pei Shi colored mould RNA | 0.01ng |
| RNA of verrucous cylinder mould | 0.001ng |
| Cryptococcus neoformans RNA | 0.001ng |
| Pneumocystis RNA | 0.001ng |
| Sporothrix scheelitis RNA | 0.001ng |
| Adding DEPC water to | 100μL |
The concentration of RNA standard was determined by Nanodrop and diluted with DEPC water to 100 ng/. Mu.L of RNA standard.
2) RNA standard library construction
100ng of RNA standard was used with the following holothurianConstruction of RNA NGS library was performed by Ultima Dual-mode RNA Library Prep Kit for Illumina (Cat # 12252). And sequenced on an Illumina NovaSeq 6000 platform.
TABLE 3 Table 3
| Component (A) | Dosage of |
| Prepared human pathogenic RNA standard | 0.1-1000ng |
| 2μM human rRNA probe mix(202110257924.X) | 1μL |
| Mu. M fungus rRNA probe mix (example 1) | 1μL |
| 2×Frag/Prime buffer | 8.5μL |
| Adding DEPC water to | 17μL |
And (5) carrying out instantaneous separation after blowing and mixing uniformly. The mixture was left at room temperature for 5min at 95℃for 1min at 75℃for 1min at 55 ℃.
TABLE 4 Table 4
| Component (A) | Dosage of |
| The above reaction system | 17μL |
| Strand Specificity Reagent | 6μL |
| 1st Strand Enzyme Mix | 2μL |
| Total volume of | 25μL |
Hieff according to the holy of the nextThe instructions of Ultima Dual-mode RNA Library Prep Kit for Illumina (Cat#12252) carry out two-strand synthesis, linker ligation and PCR amplification. After the recovered library was quantified with Qubit, the size distribution of the library was examined with Qsep. The library obtained was sequenced on a NovaSeq 6000 platform of Illumina. The sequencing results were aligned to human and the pathogenic fungus transcriptome, respectively, for analysis.
Schematic representation of RNA mNGS detection using fungal rRNA conserved region reverse transcription blocking probes is shown in FIG. 1. The results of mNGS detection on RNA standards are shown in FIGS. 2-5, and the addition of the human rRNA reverse transcription blocking probe significantly increased the fungal RNA ratio (about 8-fold) and the addition of the fungal rRNA conserved region reverse transcription blocking probe significantly increased the fungal rRNA non-conserved region and other non-rRNA data ratio (about 4.5-fold) (FIG. 2). We examined the fungus detection efficiency at different sequencing depths, and found that the addition of the human rRNA reverse transcription blocking probe and the fungus rRNA conserved region reverse transcription blocking probe can effectively reduce the sequencing depth required by fungus detection and improve the detection efficiency (FIG. 3 and FIG. 4). The three sets of data showed good linear correlation, indicating that the addition of the rapid removal probe did not cause significant loss and preference during library construction (fig. 4). These demonstrate that reverse transcription blocking probes can effectively increase the effective data duty cycle of fungal RNA and reduce the cost of fungal detection. In addition, we also tested the results of fungus detection under the condition of different input amounts of the reverse transcription blocking probe to the RNA standard, and found that the addition of the reverse transcription blocking probe in the fungus rRNA conserved region can significantly improve the sensitivity of RNA mNGS detection, and can achieve effective fungus detection of RNA as low as 0.1ng (FIG. 5).
Example 3: reverse transcription of the conserved region of fungal rRNA impedes the role of the probe in the detection of pathogenic RNA samples.
In this example, we have performed mRNA NGS detection on pathogenic sample RNAs derived from gold key medicine by a fungal rRNA conserved region reverse transcription blocking probe. The embodiment is as in example 2.
The results are shown in FIGS. 6 and 7, and the detection accuracy, efficiency and sensitivity of fungi in pathogenic samples of the probe are hindered by the addition of a human rRNA reverse transcription hindered probe and a fungal rRNA conserved region reverse transcription hindered probe.
In summary, the invention discloses a set of DNA probe compositions for preventing reverse transcription of fungal rRNA conserved regions, wherein the set of probes is designed according to 18S rRNA and 25S rRNA sequence conserved regions of 246 common fungi recorded in SILVA database, and comprises 114 probes (SEQ ID NO. 1-114), each probe can bind to not less than 10% of common fungal rRNA, can specifically and efficiently identify and bind to the conserved sequence regions of the target fungal rRNA, and prevent reverse transcription of RNA of the regions, and meanwhile retains normal reverse transcription of rRNA of non-conserved regions. The fungus rRNA reverse transcription probe disclosed by the invention has the advantages of simplicity in operation (one-step operation), short time consumption (2 min), small loss, low cost and the like, remarkably improves the effective data duty ratio, detection rate, sensitivity and accuracy of the RNA mNSS detection technology, and is very suitable for the automatic detection of the RNA mNSS.
Sequence listing
<110> next san Jose Biotech (Shanghai) stock Co., ltd
<120> probe composition for blocking reverse transcription of fungal conserved region and use thereof
<141> 2021-07-21
<160> 114
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ctggcaggat caaccag 17
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gacatgcatg gcttaatct 19
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ctgatttaat gagccattcg 20
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
agctctagaa ttaccacagt t 21
<210> 5
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
acaccccttc cgaagtcg 18
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tcatcaaaga gtccgaaga 19
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
catcgccagc acaaggcc 18
<210> 8
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gccactatcc taccatcga 19
<210> 9
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gaatcgaacc cttattccc 19
<210> 10
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gcctgctgcc ttccttgg 18
<210> 11
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ctgtatcgtt atttattgtc ac 22
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
tcgttaaggt atttacattg t 21
<210> 13
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ctggaattac cgcggctgct gg 22
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ctacgagctt tttaactgc 19
<210> 15
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
cacgaagaaa atcggac 17
<210> 16
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ggttagccag aaggaaag 18
<210> 17
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gaacactcta attttttcaa agt 23
<210> 18
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
tcctattcta ttattccatg ct 22
<210> 19
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
cattacgatg gtcctagaaa cc 22
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
ttcacctctg acaattgaat ac 22
<210> 21
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
cttggcaaat gctttcgcag t 21
<210> 22
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gtatctgatc gtcttcgatc c 21
<210> 23
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ccgatcccta gtcggca 17
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
gaacccaaag actttgattt ct 22
<210> 25
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ccttccgtca attcctttaa gtttc 25
<210> 26
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
cccgtgttga gtcaaattaa gcc 23
<210> 27
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gagctctcaa tctgtcaatc ctt 23
<210> 28
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
caccaactaa gaacggcca 19
<210> 29
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gtaggttaag gtctcgtt 18
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cgatagtccc tctaagaagt 20
<210> 31
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
ctaagggcat cacagacctg t 21
<210> 32
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
gctggctccg tcagtgta 18
<210> 33
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
agacctctcg gccaaggt 18
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
tgctctatcc ccagcacgac 20
<210> 35
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
cgcttactag gaattcct 18
<210> 36
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
ggtgtgtaca aagggcagg 19
<210> 37
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
atcctgaggc ctcactaag 19
<210> 38
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
tgaccagatt ttccgctctg a 21
<210> 39
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
cttgttacga cttttacttc ctc 23
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
tgatccttcc gcaggttcac 20
<210> 41
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gttggtttct tttcctccgc tta 23
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
tgagcttttg ccgcttcact 20
<210> 43
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ccaaggtact ttcttcaaat taca 24
<210> 44
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
cctctatgac gtcctgttcc a 21
<210> 45
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
tcgaaagaac cttagatggc act 23
<210> 46
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
gatggaattt accacccact tagag 25
<210> 47
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
ccatcactgt acttgttcgc tatc 24
<210> 48
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
cgtacttttt cactctcttt tcaa 24
<210> 49
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
cgaaggtacg actggcttca 20
<210> 50
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
cgagggccgc aaaacacc 18
<210> 51
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
cagtgaactg cggtgaccc 19
<210> 52
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
agtcacattc ctaagggttt c 21
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
ggttggcagt ataaccttag gc 22
<210> 54
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
cagacaggta gccccga 17
<210> 55
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
tgaccatcga ctttgataac c 21
<210> 56
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
ggcatttaac cattatgcca gca 23
<210> 57
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
gcatagacgt tagactcctt gg 22
<210> 58
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
cgttcacttt cattacgcgt 20
<210> 59
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
caggatcggt cgatgatgca 20
<210> 60
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
ctttcgggtc ccaacagcta tg 22
<210> 61
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
accagagttt cctctggctt c 21
<210> 62
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
cctataccca aattcgacga tcg 23
<210> 63
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
tcggcaggaa ccagctacta 20
<210> 64
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
tcgctttacc tcataaaact ga 22
<210> 65
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
gtttgagaat aggtcaagg 19
<210> 66
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
ttcattcata tgtccacgtt ca 22
<210> 67
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
cgcatcgcca gttctgctt 19
<210> 68
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
tgtctgatga gcgtgtattc 20
<210> 69
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
catggccacc gtccggct 18
<210> 70
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
gttcattcgg ccggtgagtt 20
<210> 71
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
cctgacggtg gagtataggt a 21
<210> 72
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
cactgacctc cacgcctgcc 20
<210> 73
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
accaagatct gcactagagg cc 22
<210> 74
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
cctttcccca cttcagtctt ca 22
<210> 75
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
tctcttagga tcgactaacc c 21
<210> 76
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
tcgatggtgg cctggaa 17
<210> 77
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
ccatatccag gttccggaa 19
<210> 78
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
ctcacgccga cgtctcc 17
<210> 79
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
ccaattccgg ggtgataag 19
<210> 80
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
tgctgggctc ttccagc 17
<210> 81
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
ccttcctgtg gattttcaag 20
<210> 82
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
tggagacctg ctgcggtt 18
<210> 83
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
ttgccgactt cccttatcta 20
<210> 84
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
cactacccga tccttagagc c 21
<210> 85
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 85
cgaggacaga ccacaagcac 20
<210> 86
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 86
acgacagagc acgcaagcag 20
<210> 87
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 87
tgtagcaagc gacgaccag 19
<210> 88
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 88
gacagtcaga ttccccttgt c 21
<210> 89
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
acattgcgtc aacatcactt tc 22
<210> 90
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
cgcttggttg aatttcttca c 21
<210> 91
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
cgaggcattt ggctacctta ag 22
<210> 92
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 92
gacagtggga atctcgttaa tc 22
<210> 93
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
caagcccgtt cccttggctg 20
<210> 94
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
gtcaaactag agtcaagctc a 21
<210> 95
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 95
ctggcgccga agctcccac 19
<210> 96
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 96
gcttaattga ataagtaaag aaac 24
<210> 97
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 97
ccgtatagga ctttaaatgc taga 24
<210> 98
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 98
ccagccaaac tccccacctg ac 22
<210> 99
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 99
cccttaggac atctgcgt 18
<210> 100
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 100
tcaaggggac ttttaccctt 20
<210> 101
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
aggatcgata ggccacactt tc 22
<210> 102
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 102
tggtaacttt tctggcacct ct 22
<210> 103
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 103
gcaatgtcgc tatgaacgct tg 22
<210> 104
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 104
cgaattctgc ttcggtatga tag 23
<210> 105
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 105
ctaaacccag ctcacgttcc ct 22
<210> 106
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 106
tgcgataaca ttcatcagta gg 22
<210> 107
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 107
accaattatc cgaatgaact gtt 23
<210> 108
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 108
gatggtagct tcgcggcaa 19
<210> 109
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 109
cgcgttctag catggat 17
<210> 110
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 110
gctaaaagca ccttattcgt atc 23
<210> 111
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 111
tccgtctgaa caccagttgc 20
<210> 112
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 112
ctctcgcgca gtatgacatt g 21
<210> 113
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 113
ctactgctta caataccctg ttg 23
<210> 114
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 114
tcagacaacg aaggcttaat ctc 23
Claims (3)
1. A probe composition for preventing reverse transcription of fungus 18S rRNA and 25S rRNA conserved regions, which is characterized by comprising probe mixtures shown in the following table
2. The probe composition according to claim 1, wherein the base of the italicized underlined part in the probe sequence is LNA modified base, 3' -terminal-NH 2 C 6 And (5) sealing.
3. Use of a probe composition according to claim 1 or 2 in RNA pooling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110827350.5A CN113462685B (en) | 2021-07-21 | 2021-07-21 | Probe composition for preventing reverse transcription of fungus conserved region and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110827350.5A CN113462685B (en) | 2021-07-21 | 2021-07-21 | Probe composition for preventing reverse transcription of fungus conserved region and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113462685A CN113462685A (en) | 2021-10-01 |
| CN113462685B true CN113462685B (en) | 2023-08-22 |
Family
ID=77881741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110827350.5A Active CN113462685B (en) | 2021-07-21 | 2021-07-21 | Probe composition for preventing reverse transcription of fungus conserved region and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113462685B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113862334A (en) * | 2021-10-19 | 2021-12-31 | 翌圣生物科技(上海)股份有限公司 | Probe for sealing ribosome RNA or globulin RNA in RNA library building process and application thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1274010A (en) * | 2000-03-24 | 2000-11-22 | 中山大学 | Characteristic nucleotide sequence and method for discriminating cordyceps |
| JP2001500380A (en) * | 1996-09-16 | 2001-01-16 | ザ カバメント オブ ザ ユーナイテッド ステイツ オブ アメリカ，リプリゼンテッド バイ ザ セクレタリー，デパートメント オブ ヘルス アンド ヒューマン サービシーズ，センターズ フォー ディジィーズ コントロール アンド プリベンション，テクノロジー トランスファー オフィス | Candida spp. Methods and compositions for the detection of |
| AU5422101A (en) * | 1996-11-04 | 2001-09-06 | Infectio Diagnostic (I.D.I.) Inc. | Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories |
| AU4058802A (en) * | 1994-11-09 | 2002-06-27 | Third Wave Technologies, Inc. | Rapid detection and identification of nucleic acid variants and pathogens |
| JP2004337069A (en) * | 2003-05-15 | 2004-12-02 | House Foods Corp | Method for identifying foreign substance derived from plant and primer set for use in the method |
| AU2006249236A1 (en) * | 2001-01-26 | 2007-01-04 | Intercell Ag | A Method for Identification, Isolation and Production of Antigens to a Specific Pathogen |
| CN110616253A (en) * | 2019-06-14 | 2019-12-27 | 深圳市妇幼保健院 | Kit and method for detecting fungi based on micro-drop digital PCR technology |
| CN112176031A (en) * | 2020-09-18 | 2021-01-05 | 上海英基生物科技有限公司 | Construction method and kit of enucleated ribosome RNA sequencing library |
| CN112626176A (en) * | 2021-03-10 | 2021-04-09 | 翌圣生物科技（上海）有限公司 | Reverse transcription blocking probe for quickly removing target RNA in RNA library construction and application thereof |
| CN113355750A (en) * | 2021-01-06 | 2021-09-07 | 南京诺唯赞生物科技股份有限公司 | rRNA silent RNA library construction method and kit |
| CN113564269A (en) * | 2021-07-21 | 2021-10-29 | 翌圣生物科技(上海)股份有限公司 | Probe composition for preventing reverse transcription of bacterial conserved region and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007018563A2 (en) * | 2004-10-05 | 2007-02-15 | Wyeth | Probe arrays for detecting multiple strains of different species |
| EP2370811A4 (en) * | 2008-11-26 | 2012-12-19 | Hutchinson Fred Cancer Res | Broad range pcr-based compositions and methods for the detection and identification of fungal pathogens |
-
2021
- 2021-07-21 CN CN202110827350.5A patent/CN113462685B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU4058802A (en) * | 1994-11-09 | 2002-06-27 | Third Wave Technologies, Inc. | Rapid detection and identification of nucleic acid variants and pathogens |
| JP2001500380A (en) * | 1996-09-16 | 2001-01-16 | ザ カバメント オブ ザ ユーナイテッド ステイツ オブ アメリカ，リプリゼンテッド バイ ザ セクレタリー，デパートメント オブ ヘルス アンド ヒューマン サービシーズ，センターズ フォー ディジィーズ コントロール アンド プリベンション，テクノロジー トランスファー オフィス | Candida spp. Methods and compositions for the detection of |
| AU5422101A (en) * | 1996-11-04 | 2001-09-06 | Infectio Diagnostic (I.D.I.) Inc. | Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories |
| CN1274010A (en) * | 2000-03-24 | 2000-11-22 | 中山大学 | Characteristic nucleotide sequence and method for discriminating cordyceps |
| AU2006249236A1 (en) * | 2001-01-26 | 2007-01-04 | Intercell Ag | A Method for Identification, Isolation and Production of Antigens to a Specific Pathogen |
| JP2004337069A (en) * | 2003-05-15 | 2004-12-02 | House Foods Corp | Method for identifying foreign substance derived from plant and primer set for use in the method |
| CN110616253A (en) * | 2019-06-14 | 2019-12-27 | 深圳市妇幼保健院 | Kit and method for detecting fungi based on micro-drop digital PCR technology |
| CN112176031A (en) * | 2020-09-18 | 2021-01-05 | 上海英基生物科技有限公司 | Construction method and kit of enucleated ribosome RNA sequencing library |
| CN113355750A (en) * | 2021-01-06 | 2021-09-07 | 南京诺唯赞生物科技股份有限公司 | rRNA silent RNA library construction method and kit |
| CN112626176A (en) * | 2021-03-10 | 2021-04-09 | 翌圣生物科技（上海）有限公司 | Reverse transcription blocking probe for quickly removing target RNA in RNA library construction and application thereof |
| CN113564269A (en) * | 2021-07-21 | 2021-10-29 | 翌圣生物科技(上海)股份有限公司 | Probe composition for preventing reverse transcription of bacterial conserved region and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 18S rRNA基因序列分析法鉴定临床常见酵母样真菌;耿佳靖等;《2008年微生物实用技术生态环境应用学术研讨会》;第86-94页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113462685A (en) | 2021-10-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220389416A1 (en) | COMPOSITIONS AND METHODS FOR CONSTRUCTING STRAND SPECIFIC cDNA LIBRARIES | |
| EP2989212B1 (en) | Strand-invasion based dna amplification method | |
| CN113462685B (en) | Probe composition for preventing reverse transcription of fungus conserved region and application thereof | |
| US7749707B2 (en) | Method for obtaining subtraction polynucleotide | |
| EP2092073B1 (en) | Nucleic acids probes for detection of yeast and fungal species | |
| CN110804652B (en) | Additive, kit and reaction method for real-time quantitative PCR (polymerase chain reaction) for rapidly detecting DNA (deoxyribonucleic acid) | |
| AU2011209624B2 (en) | Methods and kits used in the detection of fungus | |
| CN111961745A (en) | Method and kit for detecting multiple dermatophytes at one time | |
| US20180371523A1 (en) | Methods and materials for detecting rna sequences | |
| CN110628920A (en) | Fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof | |
| CN113564269A (en) | Probe composition for preventing reverse transcription of bacterial conserved region and application thereof | |
| US10093988B2 (en) | Universal primers and the use thereof for the detection and identification of amphibia/fish species | |
| CN114686595B (en) | SNP molecular marker for sex identification of silver dragon fish and application thereof | |
| CN114686594B (en) | SNP molecular marker suitable for sex identification of silver dragon fish and application thereof | |
| Casas Castaño et al. | Experimental design and protocols for conducting pilot studies to assess the implementation of genomic methods into fisheries research surveys | |
| CN114561477A (en) | Cattle STR composite amplification detection kit, primer composition and application thereof | |
| CN117210579A (en) | Primer group for amplifying antelope microsatellite fragment and application thereof | |
| CN114317803A (en) | Specific detection target Hpar1567 of heterobasidiomycetes and application thereof | |
| CN112011599A (en) | PCR primer set for reducing non-specific amplification and application thereof | |
| CN104328118A (en) | Hyla tsinlingensis microsatellite sites, primers for microsatellite sites and application of microsatellite sites and primers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |