AU2020101928A4 - Application and method of Imp gene in preparing kit for detecting Riemerella anatipestifer serotype - Google Patents
Application and method of Imp gene in preparing kit for detecting Riemerella anatipestifer serotype Download PDFInfo
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Abstract
The present invention relates to an application and method of IMP gene in preparing a kit for
detecting Riemerella anatipestifer serotype 1, including the following steps: designing
amplification primer pair sequences SEQ ID NO.2 and SEQ ID NO.3 according to Imp gene
sequence SEQ ID NO.1 in riemerella anatipestifer genomic DNA sequence, adding FAM
fluorescent markers at 5' and 3' ends, extracting sample DNA, and amplifying by PCR; The
PCR amplification products were recovered from the liquid of DNA recovery kit and stored
at -20°C away from light for later use; The PNA synthesized as SEQ ID NO.4 was used as
probe for reverse hybridization and observed under fluorescence microscope. The detection
method of Riemerella anatipestifer serotype 1 has the advantages of short detection time, low
cost, specific detection result and simple result judgment
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Figure 1
Description
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Figure 1
PATENTS ACT 1990
[Application and method of Imp gene in preparing kit for detecting Riemerella
anatipestifer serotype]
The invention is described in the following statement:-
Application and method of Imp gene in preparing kit for detecting
Riemerella anatipestifer serotype
The invention belongs to the field of livestock and poultry detection, and relates to a kit
for detecting Riemerella anatipestifer serotype 1 by using PNA probe reverse
hybridization, and also relates to a detection method using the kit.
Riemerella anatipestifer (RA) is the pathogen of infectious serositis in ducks, which is
one of the important diseases endangering duck industry.This disease is an acute or
chronic septicemia process, which is characterized by cellulose pericarditis, perihepatitis,
pneumonitis, meningitis, some caseous salpingitis and arthritis.The morbidity is 10%
%, and the mortality rate is as high as 80%, which can occur all year round.The disease
is prevalent in almost all duck-raising areas in the world, which has caused huge
economic losses to the duck-raising industry.Traditional identification of RA usually
detects phenotypic indicators such as culture characteristics, morphological staining,
physiological and biochemical characteristics, and some chemical compositions of
bacteria. However, the identification of Riemerella anatipestifer by phenotypic indicators
is insufficient, which is easy to be confused by Escherichia coli and Pasteurella.In
addition, the traditional identification method is complicated, time-consuming and laborious, which is not conducive to timely diagnosis of etiology, examination of pathogens and control of disease spread.According to these characteristics of Riemerella anatipestifer, many researchers have established some detection technologies, such as fluorescent antibody technology, ELLSA, immunohistochemistry and so on, but there are limitations in practice, such as the need for antibodies against different serotypes as detection reagents.In order to detect Riemerella anatipestifer serotype 1 rapidly, accurately and simply, a reverse hybridization method with PNA as probe was established.
In view of this, one of the purposes of the present invention is to provide the application
of the reagent for detecting the Imp gene of Riemerella anatipestifer in preparing the kit
for detecting Riemerella anatipestifer.The second purpose of the present invention is to
provide a kit for detecting Riemerella anatipestifer serotype 1 by PNA probe reverse
hybridization.The third purpose of the present invention is to provide a method for
detecting Riemerella anatipestifer serotype 1 by using the kit.
In order to achieve the above purpose, the present invention provides the following
technical scheme:
1. application of preparing a kit for detecting Riemerella anatipestifer serotype 1, wherein
the sequence of the Imp gene is shown in SEQ ID NO.1;And the kit is a PNA probe
reverse hybridization kit, and the kit contains a PNA probe shown in SEQ ID NO.4
And preferably, the kit further comprises a specific detection primer, wherein the 3' end
and the 5' end of the specific detection primer are modified with FAM, and the nucleotide
sequence is shown as SEQ ID NO.2 and SEQ ID No.3.
2. the kit for detecting riemerella anatipestifer serotype 1 by PNA probe reverse
hybridization comprises a specific detection primer and a PNA probe, wherein the
specific detection primer is modified with FAM at the 3' end and the 5' end, and the
nucleotide sequence is shown in SEQ ID NO.2 and SEQ ID NO.3;And the PNA probe is
shown in SEQ ID NO.4
Preferably, it also comprises Premix Taq Mix enzyme and double distilled water.
Preferably, reverse hybridization reagent is also included.
Preferably, the method for detecting Riemerella anatipestifer serotype 1 by using the kit
comprises the following steps:
(1) designing specific detection primers as shown in SEQ ID NO.2 and SEQ ID NO.3 and
PNA probes as shown in SEQ ID NO.4 according to the Imp gene sequence of
Riemerella anatipestifer, modifying FAM at the 3' end and 5' end of the detection
primers, extracting sample DNA to be detected at the same time, then using the extracted
sample DNA as a template, performing PCR amplification with the specific detection
primers as shown in SEQ ID NO.2 and SEQ ID NO.3,
(2) Fix PNA probe on activated PVDF filter membrane, hybridize in hybridization
solution containing recovered products after pre-hybridization, wash the membrane, observe under green fluorescence wavelength of fluorescence microscope, if green fluorescent spot appears in the sample, it is Riemerella anatipestifer serotype 1, if not, it is other strains.
Preferably, the condition of PCR amplification is pre-denaturation at 95C for 5
min;Denaturing at 95C for 30s, annealing at 48C for 30 s and extending at 72C for 30
s;A total of 30 cycles, extending at 72C for 10 min, and cooling to 16°C.
Preferably, the specific steps of fixing the PNA probe on the activated PVDF filter
membrane are as follows: soak the PVDF filter membrane in methanol for 15s for
activation, then it was transferred to the membrane transfer buffer and soaked for 30min,
then soak it in 6xSSC solution for 30min, and dry it at 18-25°C. After adding the PNA
probe with a concentration of 10uM to the membrane, it was dried at 18-25C for 1h,then
cross-linked by UV at 258nm for 30min,and baked at 60'C for 30min.
Preferably, the pre-hybridization is to preheat the pre-hybridization solution to 55C, then
put the membrane on which the PNA probe is fixed into it, and shake it in a water bath at
°C for 10-12 hours;The membrane washing is to rinse the hybridized membrane with
2xSSC and 0.5% SDS solution at 55C for 2 times, each time for 1h, then move the filter
membrane into 0.1xSSC and 0.1% SDS solution, shake it gently at 55C for 1h, repeat it
twice, and finally keep the temperature for 30min.
The detection method has the advantages of short detection time, low detection cost and
high accuracy in detecting Riemerella anatipestifer serum type 1.It avoids the disadvantages of complicated operation, long time-consuming, low detection rate, more false positives/false negatives, etc.At the same time, the antiserum of Riemerella anatipestifer serotype 1, which must be used in the traditional method, is not needed, thus reducing the detection cost.The detection target of the invention has single specificity, the detection result is specific, and the result judgment is simple.
In order to make the purpose, technical scheme and beneficial effects of the present
invention clearer, the present invention provides the following drawings for explanation:
Fig. 1 shows the specificity evaluation and experimental results of some clinical isolates
(1: RACH-1 (RCH);2: RA CH-2; 3: RA ATCC11845; 4: RA
RCAD0200; 5: RCAD0025; 6: RCAD0026; 7: RCAD0192; 8: RCAD0210; 9
: RCAD0155; 10: RCAD0190; 11: RCAD0195; 12: RCAD0220; 13:ATCC
25922 strains;14: Salmonella anatis CMCC 50083 strains;15: Salmonella. typhimurium
CMCC 50115 strains;16: Pasteurella. multiocida pm966 strains).
Fig. 2 shows the experimental results of sensitivity evaluation (1-11: the concentrations
of PNA probes are 50[M, 25tM, 12.5[tM, 6.2[tM, 3.1[tM, 1.6[tM, 0.8[tM, 0.4[ M,
0.2tM, 0.1 M and 0.05 m respectively;12: ddH20) o
Fig. 3 shows the detection results of clinical isolates of Riemerella
anatipestifer;RCAD135 strains is Riemerella anatipestifer serotype 1, while RCAD188
strains is not Riemerella anatipestifer serotype 1.
The present invention will be further explained with specific examples.It should be
understood that these examples are only used to illustrate the present invention and are
not used to limit the scope of the present invention.The experimental methods without
specific conditions in the following implementation are usually in accordanc with
conventional conditions such as Sambrook molecular cloning: Laboratory Manual (New
York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by
the manufacturer.
Example 1: establishment of a method for detecting Riemerella anatipestifer serotype 1
by PNA probe reverse hybridization
Primer pairs for specific amplification of Riemerella anatipestifer and PNA probes for
specific hybridization of Riemerella anatipestifer serotype 1 were designed: through
bioinformatics analysis, Imp gene was found from the genomic DNA sequence of
Riemerella anatipestifer. Imp is highly conserved among other Gram-negative bacteria
and has many important biological functions, besides participating in the process of
lipopolysaccharide export and assembly to the outer membrane, it is also related to the
formation of bacterial envelope and the tolerance of bacteria to organic solvents.It can be used as the detection target gene of Riemerella anatipestifer serotype 1, and the gene sequence is shown in SEQ ID NO.1
(1) design of primer and PNA probe
According to the Imp gene sequence, primers with the size range of 100-500bp and PNA
probes specific to Riemerella anatipestifer serotype 1 in PCR amplification products were
designed.,the specific sequences are as follows (primers were synthesized by Chengdu
qingke zixi biotechnology co., ltd.):
Upstream primer: 5' fam-aaaagaaatgacttacct-fam3' (SEQ ID NO.2);
Downstream primer: 5' fam-tacatttgtataggtcctg-fam3' (SEQ ID NO.3);
Sequence of PNA: N terminal-ccccttgcga-c terminal (SEQ ID NO.4), synthesized by
panagene company.
(2) Preparation of DNA template
The strains of different serotypes of Riemerella anatipestifer were inoculated into 10ml of
pancreatic enzyme soy broth liquid culture medium, and after 12 hours of enrichment at
37°C, 1ml of bacteria solution was put into a 1.5ml centrifuge tube, centrifuged at 3000
r/min for 10mmin, and then the supernatant was discarded and resuspended in sterile
double distilled water, then centrifuged at 12000 r/min for 5min, and the supernatant was
discarded and the bacteria were collected.resuspension of bacteria with sterile double
distilled water.Boiling in boiling water bath for 15 minutes, taking out immediately, and standing at -20°C for 30 min; ;Thawing at 37C, centrifuging at 12000 r/min for 5min, taking supernatant and placing it at -20°C for later use.
(3) PCR amplification
PCR amplification system is a 25pL reaction system, specifically: Premix Taq Mix
enzyme 12.5t, 10tM primer pair 1.0 d, template solution 1-2.5 d, and finally added to
1 with double distilled water. PCR detection reaction procedure: pre-denature at 95°C
for 5min, then start amplification cycle, and the procedure of each cycle is: denature at 95
°C for 30 s, anneal at 48C for 30s, and extend at 72C for 30s;After 30 cycles, extend at
72°C for 10 min, and cool to 16°C.
(4) PCR product recovery and probe fixation
The PCR product liquid was recovered by DNA recovery kit, and stored at -20°C away
from light for later use.Immobilization of probe: PVDF filter membrane (pore size:
0.22[tm) was soaked in methanol for 15s for activation, then moved to membrane transfer
buffer for 30min, then soaked in 6xSSC solution for 30min, and dried at room
temperature. After adding 10jM PNA probe to the membrane, it was dried at room
temperature for 1h, and then UV-linked at 258nm for 30min and baked at 60°C for
min.
(5) Pre-hybridization
Prehybridization solution preheated to 55°C (6xSSC, 0.5%SDS, 5xDenhardt solution,
100pg/ml salmon sperm DNA. The 6xSSC components are as follows: 52.5g/L sodium
chloride, 26.5g/L trisodium citrate), put the membrane into it, and shake it in a water bath
at 55C for 10-12 hours. Water bubbles often form on the surface of the membrane, and
these small bubbles can be removed by gently shaking the hybridization solution, which
is very important to ensure that the surface of the membrane is fully saturated with the
prehybridization solution.
(6) Hybridization
Preheat the hybridization solution to 60°C (insert 2 1 of DNA recovered from PCR
products into the prehybridization solution), transfer the membrane into it, and water bath
at 60°C for 12h;
(7) membrane washing
Rinse with 2xSSC and 0.5%SDS solution at 55C for lhx2, then move the filter
membrane into 0.1%xSSC and 0.1%xSDS solution, shake gently at 55C for lhx2, and
finally keep the temperature for 30min;
(8) taking pictures
Under the green fluorescence wavelength of fluorescence microscope, it is Riemerella
anatipestifer serotype 1 if there is green fluorescent spot in the sample, and other strains if
there is no green fluorescent spot.
Results: Under the fluorescence microscope, only the serotype 1 of Riemerella
anatipestifer showed green fluorescent spots.
Example 2 specificity evaluation experiment of PNA reverse hybridization detection
method
Escherichia coli, Pasteurella multocida, Salmonella and Riemerella anatipestifer strains of
various serotypes (preserved strains, clinical isolates of Riemerella anatipestifer of
unknown serotype or serotype identified by serological test, as shown in Table 1) were
detected according to the DNA template extraction method and PNA probe reverse
hybridization detection method respectively.
The detection results of PNA probe reverse hybridization specificity experiment are
shown in Figure 1 and Table 1, and only Riemerella anatipestifer serotype 1 appears
green fluorescent spots.
Table 1. Strains and test results used for specific evaluation of PNA probe reverse
hybridization
Strain number Clinical isolates with Strain name and serotype Detection result preservation number or identification
RACH-1(RCH) CCTCC NO.M2017702 Serotype 1 of Riemerella +
anatipestifer
RA CH-2 Identification of clinical Riemerella anatipestifer isolates of serotypes serotype 2
RCADO190 Identification of clinical Riemerella anatipestifer - isolates of serotypes serotype 2
RAATCC11845 ATCC11845 Riemerellaanatipestifer serotype 6
RCADO192 Identification of clinical Riemerella anatipestifer isolates of serotypes serotype 11
RCAD0025 Identified clinical isolates Riemerella anatipestifer of unknown serotype
RCAD0026 Identified clinical isolates Riemerella anatipestifer of unknown serotype
RCADO155 Identified clinical isolates Riemerella anatipestifer of unknown serotype
RCADO195 Identification of clinical Riemerella anatipestifer isolates of serotypes serotype 16
RCAD0200 Identified clinical isolates Riemerella anatipestifer of unknown serotype
RCAD0210 NTCC698316 Escherichia coli X7213
RCAD0220 NTCC503720 Escherichia coli XL1-blue
E.coli 06 ATCC25922 Escherichia coli 06 serotype
Salmonella anatis Salmonella CMCC 50083 Salmonella duck 50083 strains
Salmonella.typhimurium CMCC(B)50115 Salmonella typhimurium 50115
Pasteurella multiocida PM966 Pasteurella multocida PM966
In Table 1, -: PNA probe reverse hybridization results are negative: +: PNA probe reverse
hybridization results are positive.
Example 3 sensitivity evaluation experiment for detection of riemerella anatipestifer
serotype 1 by PNA reverse hybridization
The initial concentration of PNA probe is 50[M, which is diluted with sterile water twice
in gradient, with a total of 10 gradients. 21l of each gradient is used for probe fixation,
and reverse hybridization detection is carried out according to the steps of Example 1, as
shown in Figure 2.It can be seen from fig 2 that fig 5 (corresponding PNA probe
concentration is 3.1pM) can see fluorescent spots, and fig 4 (corresponding PNA probe
concentration is 6.2pM) can see clear fluorescent spots with extremely high sensitivity.
Example 4 Detection of Clinical Isolates of Riemerella anatipestifer
100 clinical isolates of Riemerella anatipestifer isolated from ducks in more than 20
large-scale farms around Sichuan were detected by using the PNA probe reverse
hybridization detection method established in Example 1, including serotype 1, serotype
2, serotype 6, serotype 11 and other unknown serotypes.At the same time, the serum
agglutination test was carried out to compare the results of the two tests.
Results: Only RCAD135 strain showed obvious fluorescent spots (as shown in Figure 3),
while other strains had no obvious reaction. At the same time, the serum agglutination
test of 100 RA clinical isolates showed that only RCAD135 strain was serum type 1,
which was consistent with the established PNA hybridization test results. At the same
time, the sequencing results of PCR products also showed that RCAD135 strain
contained the target sequence of Riemerella anatipestifer serotype 1 under the action of
PNA, which indicated that this method could be used to detect Riemerella anatipestifer
serotype 1.
Finally, the above preferred embodiments are only used to illustrate the technical scheme
of the present invention, but not to limit it. Although the present invention has been
described in detail through the above preferred embodiments, those skilled in the art
should understand that various changes can be made in form and detail without departing
from the scope defined by the claims of the present invention.
Claims (10)
1.The application of a reagent for detecting riemerella anatipestifer Imp gene in preparing
a kit for detecting riemerella anatipestifer serotype 1 is characterized in that the sequence
of the Imp gene is shown as SEQ ID NO.1;And the kit is a PNA probe reverse
hybridization kit, and the kit contains a PNA probe shown in SEQ ID NO.4.
2.The application according to claim 1, which is characterized in that the kit also contains
specific detection primers, wherein the 3' end and 5' end of the specific detection primers
are modified with FAM, and the nucleotide sequences are shown in SEQ ID NO.2 and
seq id No.3.
3.The reagent kit for detecting riemerella anatipestifer serotype 1 by PNA probe reverse
hybridization comprises a specific detection primer and a PNA probe, wherein the
specific detection primer is modified with FAM at the 3' end and the 5' end, and the
nucleotide sequence is shown as SEQ ID NO.2 and SEQ ID NO.3;And the PNA probe is
shown in SEQ ID NO.4
4.The kit according to claim 3, which is characterized by further comprising Premix Taq
Mix enzyme and double distilled water.
5. The kit according to claim 3, which is characterized in that the reverse hybridization
reagent is also included.
6. The kit according to claim 5, characterized in that the reverse hybridization reagent
comprises PVDF filter membrane, methanol, membrane transfer buffer, 6xSSC solution, pre-hybridization solution and 0.5%SDS solution;The material components of the prehybridization solution are as follows: 6xSSC, 0.5% SDS, 5xDenhardt solution and
100 g/ml salmon sperm DNA.
7. The method for detecting Riemerella anatipestifer serotype 1 by using the kit according
to any one of claims 3-6, which is characterized by comprising the following steps:
(1) designing specific detection primers as shown in SEQ ID NO.2 and SEQ ID NO.3 and
PNA probes as shown in SEQ ID NO.4 according to the Imp gene sequence of
Riemerella anatipestifer, modifying FAM at the 3' end and 5' end of the detection
primers, extracting sample DNA to be detected at the same time, then using the extracted
sample DNA as a template, performing PCR amplification with the specific detection
primers as shown in SEQ ID NO.2 and SEQ ID NO.3, and carrying out PCR
amplification
(2) Fix PNA probe on activated PVDF filter membrane, hybridize in hybridization
solution containing recovered products after pre-hybridization, wash the membrane,
observe under green fluorescence wavelength of fluorescence microscope, if green
fluorescent spot appears in the sample, it is Riemerella anatipestifer serotype 1, if not, it
is other strains.
8. The method according to claim 7, characterized in that the PCR amplification
condition is pre-denaturation at 95C for 5 minutes;Denaturing at 95C for 30s,
annealing at 48C for 30 s, and extending at 72C for 30 s, a total of 30 cycles;Stretching
at 72C for 10 min and cooling to 16°C.
9. The method according to claim 7, wherein the specific steps of fixing the PNA probe
on the activated PVDF filter membrane are as follows: soaking the PVDF filter
membrane in methanol for 15s for activation, then moving to the membrane transfer
buffer for soaking for 30min, then fully soaking in 6xSSC solution for 30min, drying at
18-25°C, adding the PNA probe with a concentration of 1 OM to the membrane, dried at
18-25°C for lh,then cross-linked by UV at 258nm for 30min,and baked at 60°C for
min.
10. The method according to claim 7, characterized in that the pre-hybridization is to
preheat the pre-hybridization solution to 55C, then put the PNA probe-immobilized
membrane into it, and shake it in a water bath at 55C for 10-12 hours;The hybridization
is to water bath at 60°C for 12 h in hybridization solution containing PCR product
recovered DNA;The membrane washing is that the hybridized filter membrane is rinsed
twice with 2xSSC and 0.5% SDS solution at 55C for 1h each time, then the filter
membrane is moved into O.1xSSC and 0.1% SDS solution at 55C, gently shaken for 1h,
repeated twice, and finally kept for 30min.
-1/3-
Figure 1
-2/3-
Figure 2
-3/3-
Figure 3
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