CN102978282A - Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology and medical science, and provides a typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit. The detection kit comprises a first reaction solution, a second reaction solution and paraffin, wherein the first reaction solution comprises a reaction buffer solution, a primer pair and an ecprobe aiming at typhoid fever salmonella, a primer pair and an ecprobe aiming at the salmonella paratyphi. The sequence of the primer pair aiming at the typhoid fever is SEQ ID NO: 1 and SEQ ID NO: 2, the sequence of the ecprobe aiming at the typhoid fever is SEQ ID NO: 3, the sequence of the primer pair aiming at the salmonella paratyphi is SEQ ID NO: 3 and SEQ ID NO: 3, and the sequence of the ecprobe aiming at the salmonella paratyphi is SEQ ID NO: 6. The second reaction solution comprises deoxyribonucleic acid (DNA) polymerase O. The invention further provides an application of the detection kit in detection of the typhoid fever salmonella and salmonella paratyphi. According to the detection kit, the detection time is shortened, cross contamination among samples is avoided, and the result is accurate and reliable.

Description

Typhoid fever, paratyphosus A bacillus fluorescent quantificationally PCR detecting kit and application thereof

Technical field

The invention belongs to biotechnology and medical field, relate in particular to a kind of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit and application thereof.

Background technology

Typhoid fever (typhoid fever) is the acute infectious disease that is caused by Corynebacterium diphtheriae, and to continue microbemia, reticuloendothelial system is got involved, and ileum far-end microabscess and ulceration are the basic pathology feature.Paratyphoid is the acute infectious disease of the digestive tract that first, second, Bacillus paratyphosus C cause.Can be because of water source and food pollution generation eruption and prevalence.Typhoid and paratyphoid all are the legal Category B notifiable disease of China, also are one of global main public health problems.Their cause of disease Salmonella typhi and paratyphosus A bacillus are the enterobacteriaceae Salmonellas, and gram's staining is negative, is rod-short, long 1～3.5 μ m, and wide 0.5～0.8 μ m, all amphitrichous can be movable, do not produce the brood cell, without pod membrane.Amphimicrobian, the suitableeest growth temperature are 35 ~ 37 ℃, Ph6.8 ~ 7.8.They are less demanding to nutrition, and ordinary culture medium can grow the bacterium colony of circle, smooth, moistening, translucent neat in edge, but salmonella after manually going down to posterity can from smooth colony progressively transition be rough type bacterium colony.The nonfermented lactose, the bacterium colony center that forms at the SS agar plate is black.

The diagnostic method of the paratyphoid that the typhoid fever that causes for Salmonella typhi at present and paratyphosus A bacillus cause comprises:

1. blood routine examination: leukocyte count is generally in (3 ~ 5) * 10 ⁹/ L, neutrophil leucocyte reduces, and eosinophilic granulocyte reduces or disappears.Whether eosinophil count is recovered with sb.'s illness took a favorable turn normally, and the diagnosis of typhoid fever and state of an illness assessment are had some reference value, have Corynebacterium diphtheriae and paratyphosum Bacterium to exist but can not detect.

2. Bacteria culturing: hemoculture is the most frequently used foundation of making a definite diagnosis.The positive rate the highest (80% ~ 90%) in the 1st ~ 2 week of the typhoid fever course of disease is difficult for detecting after being about for 50%, the 4 week the 3rd week.Can be used as the foundation of routine inspection.

Fertilizer reach (Widal) reaction about 1 week antibody is appearring usually after being ill, the positive rate in the 3rd ~ 4 week can reach more than 70%, it is also higher to tire, and can keep the several months.Have the small number of patients antibody positive to occur later, perhaps the antibody titer level is lower.There is approximately 10% ~ 30% patient's Widal reaction to be always negative.OAg is Salmonella typhi

4. with the common antigen of paratyphoid A, second, the third bacillus, detect the high-titer OAb in the serum and can not distinguish four kinds of different pathogenic bacterial infections, but four flagellar antigen (" H ", " A ", " B ", " C ") difference can rise to judge from four specific antibody titres the bacterial classification of infection.To without the immune, the agglutination titer of OAb at 1:160 or when above, can be defined as the positive at 1:80 and " H ", " A ", " B ", " C " antibody, has auxiliary diagnosis to be worth.By reinspection in per 5 ~ 7 days 1 time, rise gradually if tire, be worth larger.If only have OAb to rise, and " H ", " A ", " B ", " C " antibody does not rise, and may be that morbidity is early stage; If opposite, only have " H ", " A ", " B ", " C " antibody to rise and OAb not increase may be non-specific anamnestic reaction because of due to other heat generation illness.Salmonella D group and A group have the common antigen of part, and the latter's infection can produce the cross reaction of " O " and " H " antibody.

5. other inspection: the amynologic diagnostic method that some are new comprises the PCR detection technique.Conventional PCR detects has quick, sensitive advantage with respect to traditional culture method, but it is unstable also to produce effect in the routine clinical application, the problems such as crossed contamination occur easily.

Summary of the invention

The purpose of the embodiment of the invention is to provide a kind of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit, is intended to solve in the prior art cycle length, complex operation, the low problem of sensitivity that detect for Salmonella typhi and paratyphosus A bacillus.

The embodiment of the invention is achieved in that a kind of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit, comprises the first reaction solution, the second reaction solution and paraffin, and this first reaction solution comprises: reaction buffer; For the primer of Salmonella typhi to and specific probe, this primer is SEQ IDNO:1 and SEQ ID NO:2 to sequence, this specific probe sequence is SEQ ID NO:3, for the primer of paratyphosus A bacillus to and specific probe, this primer is SEQ ID NO:4 and SEQ IDNO:5 to sequence, this specific probe sequence is SEQ ID NO:6, and this second reaction solution comprises archaeal dna polymerase O.

Another purpose of the embodiment of the invention is to provide Salmonella typhi of the present invention and the application of paratyphosus A bacillus fluorescent quantificationally PCR detecting kit in Salmonella typhi and paratyphosus A bacillus detection.

Salmonella typhi of the present invention and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit utilize fluorescent quantitative PCR technique, have avoided the deficiencies such as operational cycle length, complex steps in the culture-based method, have the quick and precisely advantage such as sensitivity height.And test kit of the present invention can directly utilize blood sample to detect in the application process that is used for Salmonella typhi and paratyphosus A bacillus, do not need DNA extraction, shortened detection time, avoids crossed contamination between sample, and the result accurately and reliably.

Description of drawings

Fig. 1 is fluorescence quantitative PCR detection result schematic diagram in the prior art;

Fig. 2 is that the PCR of the test kit of the embodiment of the invention detects positive findings judgement synoptic diagram;

Fig. 3 is that the PCR of the test kit of the embodiment of the invention detects negative findings judgement synoptic diagram;

Fig. 4 is that the PCR of the embodiment of the invention 3 detects positive findings;

Fig. 5 is that the PCR of the embodiment of the invention 3 detects negative findings.

Embodiment

In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.

The embodiment of the invention provides a kind of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit, comprises the first reaction solution, the second reaction solution and paraffin, and this first reaction solution comprises: reaction buffer; For the primer of Salmonella typhi to and specific probe, this primer is SEQ ID NO:1 and SEQ ID NO:2 to sequence, this specific probe sequence is SEQ ID NO:3; For the primer of paratyphosus A bacillus to and specific probe, this primer is SEQ ID NO:4 and SEQ ID NO:5 to sequence, this specific probe sequence is SEQ ID NO:6; This second reaction solution comprises archaeal dna polymerase O.

The embodiment of the invention is utilized fluorescent quantitative PCR technique, and its ultimate principle is: introduce a kind of fluorescence chemical material in the PCR reaction system, along with the carrying out of PCR reaction, the PCR reaction product constantly accumulates, and fluorescence signal intensity also equal proportion increases.Every through a circulation, collect a fluorescence signal intensity, so just can be by the variation of fluorescence intensity variation monitoring product amount, obtain an amplified fluorescence graphic representation, as shown in Figure 1, the cycle number that the Ct value experiences when arriving the thresholding of setting for the fluorescent signal in each PCR reaction tubes, and in the curve index amplification stage, there is linear relationship between the logarithmic value of PCR product amount and the starting template amount, can carries out accordingly quantitative analysis.

Particularly, in the test kit of the embodiment of the invention, the reaction buffer that comprises in the first reaction solution contains the MgCl of 25mM ₂, do not contain 10 * buffer, the dNTPs of magnesium ion.Wherein dNTP is deoxy-ribonucleoside triphosphate(deoxyribonucleoside triphosphate) abbreviation, dNTP comprises dATP, dGTP, dTTP, dCTP in embodiments of the present invention, is used for the raw material as the PCR reaction.

Particularly, in the test kit of the embodiment of the invention, in the first reaction solution for the primer of Salmonella typhi to SEQ ID NO:1 and SEQ ID NO:2 and specific probe SEQ ID NO:3, and as shown in table 1 to SEQ ID NO:4 and SEQ ID NO:5 and specific probe SEQ IDNO:6 sequence for the primer of paratyphosus A bacillus:

Table 1 for the primer of Salmonella typhi, paratyphosus A bacillus to and specific probe sequence

Particularly, be connected with fluorophor for the specific probe SEQ ID NO:3 of Salmonella typhi with for the 5 ' end of the specific probe SEQ ID NO:6 of paratyphosus A bacillus, 3 ' end is connected with quenching group.Fluorophor can send fluorescent signal, the fluorescent signal that quenching group can the Fluorophotometry group sends, and in the quantitative fluorescent PCR reaction process, quenching group separates with fluorophor, fluorophor can send fluorescent signal, can carry out quantitative analysis to unknown template by the accumulation that detects fluorescent signal.Wherein, the fluorophor that is connected in 5 ' end can be FAM or HEX, wherein FAM is Carboxyfluorescein, Chinese is Fluoresceincarboxylic acid, HEX is hexachloro-fluorescein, Chinese is the chlordene fluorescein, this quenching group can be DABCYL or BHQ, DABCYL is 4,4-Dimethylamino-azobenzene-4'-carboxylic acid, Chinese is 4-dimethylamine nitrogen benzide-4 '-carboxylic acid, and BHQ is Black Hole Quencher, and Chinese is the black hole quencher.

Particularly, in the test kit of the embodiment of the invention, to SEQ ID NO:1 and SEQ ID NO:2 and specific probe SEQ ID NO:3, and be 45-55 μ M for the primer of paratyphosus A bacillus to SEQ ID NO:4 and SEQ ID NO:5 and the concentration of specific probe SEQ ID NO:6 in storage liquid for the primer of Salmonella typhi.Preferably, described concentration is 50 μ M.In use, this storage liquid is mixed with other components in the first reaction solution make the first reaction solution.

Particularly, in the test kit of the embodiment of the invention, also contain the sterilization ultrapure water in the first reaction solution, be used for when the test kit that uses the embodiment of the invention carries out the PCR reaction, supplying the volume of reaction system, the PCR reaction is carried out effectively.

Particularly, in the test kit of the embodiment of the invention, the archaeal dna polymerase O that contains in the second reaction solution, be Omni Klentaq, this archaeal dna polymerase is through the Taq of engineered mistake enzyme, and it lacks 5 ' → 3 ' 5 prime excision enzyme activity, can tolerate the inhibitor that exists in whole blood, the soil sample, can directly increase in the whole blood sample DNA and need not prior purification, therefore use archaeal dna polymerase O can improve the efficient that blood sample PCR detects.The concentration of this archaeal dna polymerase O in storage liquid is 40-44U/ μ L, and preferred concentration is 42U/ μ L.When using the test kit of the embodiment of the invention, this storage liquid is mixed with other components and make the second reaction solution.

Preferably, also comprise nitrite ion in the second reaction solution of the test kit of the embodiment of the invention, this nitrite ion is preferably tetrabromophenol sulfonphthalein, and nitrite ion can play the effect of indication when application of sample.

Paraffin plays the effect of isolation in the test kit of the embodiment of the invention, and the first reaction solution and the second reaction solution are separated, and is admixed together after the heating in the PCR instrument, and with the raising reaction efficiency, and this paraffin can not affect detected result.

Particularly, in the test kit of the embodiment of the invention, the volume that each reaction system does not comprise testing sample is 20 μ L, for those skilled in the art, can proportionally enlarge, for example be extended to 50 μ L, this reaction system does not comprise the paraffin that plays buffer action, and the volume of paraffin is 20 μ L.

Preferably, each reaction system in the reactor is prepared by table 2.

Table 2 reaction solution component

Preferably, during the test kit of the preparation embodiment of the invention, add first the first reaction solution in the PCR reaction tubes, mixing adds liquid paraffin, solidifies rear adding the second reaction solution until it, and paraffin is kept apart the first reaction solution and the second reaction solution thus.

The embodiment of the invention also provides the application of test kit of the present invention in Salmonella typhi and paratyphosus A bacillus detection.Particularly, when Salmonella typhi and paratyphosus A bacillus detection, get human vein blood, add 5 μ L venous blood in each reaction system of test kit of the embodiment of the invention, carry out the PCR reaction, concrete reaction conditions is:

50-55℃：2-5min；

94-95℃：2-3min；

94-95 ℃: 5-10s, 55-60 ℃: 40-80s; 40 circulations

Reaction finishes rear according to amplification collection of illustrative plates result of determination, referring to Fig. 2, Fig. 3, wherein

Positive findings: amplification curve diagram Ct value≤35, as shown in Figure 2;

Negative findings: amplification curve diagram Ct value＞35 or without Ct value (Fig. 3).

Below in conjunction with specific embodiment Salmonella typhi of the present invention and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit and application thereof are described further.

Embodiment 1

The preparation of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit.

In the PCR reaction tubes, prepare the first reaction solution of the present invention by following component:

Adding the sterilization ultrapure water makes cumulative volume reach 18.0 microlitres

Wherein, the 5 ' end of above-mentioned probe 117A (SEQ ID NO:3) is connected with FAM, and 3 ' end is connected with DABCYL; 5 ' the end of above-mentioned probe 118H (SEQ ID NO:6) is connected with HEX, and 3 ' end is connected with DABCYL.

The first reaction solution is mixed, then add paraffin 20 microlitres that melt on liquid level, after it solidified, this paraffin sealed the first following reaction solution, then adds the second reaction solution by following component proportion:

Archaeal dna polymerase O(42U/ μ L) 0.2 microlitre

Bromjophenol blue 0.01 microlitre

Then add the sterilization ultrapure water and complement to 2.0 microlitres, mix.

Embodiment 2

The preparation of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit.

In the PCR reaction tubes, prepare the first reaction solution of the present invention by following component:

Adding the sterilization ultrapure water makes cumulative volume reach 18.0 microlitres.

Wherein, the 5 ' end of above-mentioned probe 117A (SEQ ID NO:3) is connected with HEX, and 3 ' end is connected with BHQ; 5 ' the end of above-mentioned probe 118H (SEQ ID NO:6) is connected with FAM, and 3 ' end is connected with BHQ.

The first reaction solution is mixed, then add paraffin 20 microlitres that melt on liquid level, after it solidified, this paraffin sealed the first following reaction solution, then adds the second reaction solution by following component proportion:

Archaeal dna polymerase O(42U/ μ L) 0.5 microlitre

Bromjophenol blue 0.02 microlitre

Then add the sterilization ultrapure water and complement to 2.0 microlitres, mix.

Embodiment 3

The application of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit.

1. blood specimen collection

For the first all aseptic collection venous blood 5mL/ people of 20 routine typhoid patients in the course of disease.

2. real-time fluorescence quantitative PCR detects

The venous blood that step 1 is gathered is got in the PCR reaction tubes of PCR detection kit that 5 μ L directly are added to embodiment 1 preparation, this reaction tubes contains the first reaction solution and the second reaction solution, they separate with paraffin, then close the lid, this PCR reaction tubes is put into quantitative real time PCR Instrument carry out the PCR reaction.Wherein the PCR reaction conditions is:

50℃：2min；

94℃：2min；

94 ℃: 5s, 55 ℃: 40s; 40 circulations.

Reaction obtains reaction result after finishing.

3. the result judges

According to amplification collection of illustrative plates result of determination, wherein:

Positive findings: amplification curve diagram Ct value≤35, and obvious exponential growth is arranged, referring to Fig. 2.

Negative findings: amplification curve diagram Ct value＞35(is not shown) or without Ct value (see figure 3).

Concrete detected result sees Table 3, Fig. 4 and Fig. 5, and wherein Fig. 4 is that PCR detects the positive findings graphic representation, and Fig. 5 is that PCR detects the negative findings graphic representation.

The comparative example

With traditional culture method embodiment as a comparison, to detect the reference of effect as test kit of the present invention.

1. blood specimen collection

For the first all aseptic collection venous blood 10mL/ people of 20 routine typhoid patients in the course of disease.

2. increasing bacterium cultivates

Volume ratio by 1:10 is added to blood enrichment medium with blood sample, places 36 ℃ ± 1 ℃ to hatch, and turns clock blood agar plate or nutrient agar plate respectively at 1 day, 2 days, 7 days, puts 36 ℃ ± 1 ℃ and cultivates 24 hours～48 hours.

3. biochemical identification

Select from above-mentioned flat board and to be no less than 3 suspicious bacterium colonies, transferred species is to following substratum respectively: semi-solid each one of disaccharide iron inclined-plane (KI) or triple sugariron inclined-plane (TSI) and power-indole-urea, cultivated 18 hours the observation biochemical reaction for 36 ℃ ± 1 ℃.Do slide agglutination test with multivalence O serum, as present strong agglutination reaction, it is positive to be test, namely contains detected sample.

Interpretation of result

With embodiment 3(referring to Fig. 4 and Fig. 5) compare with its comparative example's detected result, obtain detected result as shown in table 3.

Table 3 detected result

Embodiment 4

The application of Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit.

1. blood specimen collection

For the first all aseptic collection venous blood 5mL/ people of 15 routine typhoid patients in the course of disease.

2. real-time fluorescence PCR detects

The venous blood that step 1 is gathered is got in the PCR reaction tubes of PCR detection kit that 5 μ L directly are added to embodiment 2 preparations, closes the lid, and this PCR reaction tubes is put into quantitative real time PCR Instrument carry out the PCR reaction.Wherein the PCR reaction conditions is:

55℃：5min；

95℃：3min；

95 ℃: 10s, 60 ℃: 80s; 40 circulations.

3. the result judges

According to amplification collection of illustrative plates result of determination, see Fig. 2, Fig. 3.

Positive findings: amplification curve diagram Ct value≤35, and obvious exponential growth is arranged.

Negative findings: amplification curve diagram Ct value＞35 or without the Ct value.

Concrete detected result sees Table 4.

The comparative example

1. blood specimen collection

For the first all aseptic collection venous blood 5mL/ people of 15 routine typhoid patients in the course of disease.

All the other operation stepss are with the comparative example of embodiment 3.

Embodiment 4 and comparative example's thereof interpretation of result sees Table 4

Table 4 detected result

According to table 3 and table 4 as can be known, the culture-based method detected result is consistent with the fluorescence quantitative PCR detection method result who adopts test kit of the present invention, and the real-time fluorescence PCR method detects and only needs about 5 hours, cultivates the rule needs more than 66 hours.

The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. a Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit comprise the first reaction solution, the second reaction solution and paraffin, and described the first reaction solution comprises: reaction buffer; For the primer of Salmonella typhi to and specific probe, described primer for Salmonella typhi is SEQ IDNO:1 and SEQ ID NO:2 to sequence, described specific probe sequence for Salmonella typhi is SEQ ID NO:3; And for the primer of paratyphosus A bacillus to and specific probe, described primer for paratyphosus A bacillus is SEQ ID NO:4 and SEQ ID NO:5 to sequence, described specific probe sequence for paratyphosus A bacillus is SEQ ID NO:6, and described the second reaction solution comprises archaeal dna polymerase O.

2. Salmonella typhi as claimed in claim 1 and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit, it is characterized in that, described for Salmonella typhi specific probe SEQ ID NO:3 and be connected with FAM or HEX for the 5 ' end of the specific probe SEQ ID NO:6 of paratyphosus A bacillus, 3 ' end is connected with DABCYL or BHQ.

3. Salmonella typhi as claimed in claim 1 and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit is characterized in that, described reaction buffer contains the MgCl of 25mM ₂, do not contain 10 * buffer and the dNTPs of magnesium ion.

4. Salmonella typhi as claimed in claim 1 and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit is characterized in that, described the second reaction solution also comprises nitrite ion.

5. Salmonella typhi as claimed in claim 1 and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit is characterized in that, described the first reaction solution and described the second reaction solution are separated by described paraffin.

6. Salmonella typhi as claimed in claim 1 and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit, it is characterized in that, described primer for Salmonella typhi is to SEQ ID NO:1 and SEQ IDNO:2, described specific probe sequence SEQ ID NO:3 for Salmonella typhi, and for the primer of paratyphosus A bacillus to SEQ ID NO:4 and SEQ ID NO:5, described specific probe sequence SEQ ID NO:6 concentration before mixing with other components in described the first reaction solution for paratyphosus A bacillus is 45-55 μ M.

7. Salmonella typhi as claimed in claim 1 and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit is characterized in that, described archaeal dna polymerase O is being 40-44U/ μ L with concentration before other components of described the second reaction solution are mixed.

8. such as the application in Salmonella typhi and paratyphosus A bacillus detection of each described Salmonella typhi and paratyphosus A bacillus fluorescent quantificationally PCR detecting kit in the claim 1 to 7.

9. application as claimed in claim 8 is characterized in that, described Salmonella typhi and paratyphosus A bacillus detect and comprise the PCR reaction, and described PCR reaction conditions is:

50-55℃：2-5min；

94-95℃：2-3min；

94-95 ℃: 5-10s, 55-60 ℃: 40-80s; 40 circulations.

10. application as claimed in claim 8 is characterized in that, described Salmonella typhi and paratyphosus A bacillus detect and comprise the step of utilizing whole blood directly to carry out the PCR reaction.