CN111537713B - Peste des petits ruminants virus antibody detection kit - Google Patents

Abstract

The invention relates to a peste des petits ruminants virus antibody detection kit. The detection kit comprises one or more solid supports, and a specific polypeptide or a specific polypeptide combination independently connected to the one or more solid supports.

Description

Peste des petits ruminants virus antibody detection kit

The application is a divisional application of an invention patent application with the application number of 201810310284.2 (the name of the invention: a peste des petits ruminants virus antibody detection kit, the application date: 2018, 04, 03).

Technical Field

The invention mainly relates to a diagnostic kit and a diagnostic method for animals. In particular, the invention relates to a kit for detecting the presence or absence of antibodies against peste des petits ruminants virus in a biological sample of biological origin from a subject.

Background

Peste des petits ruminants (PPR) is commonly called sheep plague, also called peste des petits ruminants pseudocattle plague (pseudoplague), pneumonia and stomatitis-pneumonia complex (stomatitis-pneumoenteritis complex), is an acute viral infectious disease caused by peste des ruminants virus, mainly infects peste ruminants, and is characterized by fever, stomatitis, diarrhea and pneumonia.

The disease occurs on the coast of ivory for the first time in 1942, and then, the disease is reported in Saibogal, ganna, dougo, beining and the like in Africa, and the disease also occurs in sheep and goats in Nigeria and causes great loss. This disease has also been reported in several countries in asia, and according to the world animal health Organization (OIE) report in 1993 "world animal health", goats in bangladesh have developed this disease, while sheep in parts of indian dela and maharashtra have developed a disease similar to cattle plague, and finally have been diagnosed as peste des petits ruminants, after which infection has also been reported in tamierra. In 1993, peste des petits ruminants were reported for the first time in Israel, the source of infection was unknown, and to prevent the spread of the disease, israel inoculated cattle plague vaccines to sheep and goats in northern regions. In 1992, this disease-specific antibody was found in jordan sheep and goats, and in 1993, 11 clinical cases occurred in farms, and more than 100 sheep and goats died. In 1993, 133 cases were first discovered by saudi arabia. In 2007 in 7 months, the epidemic disease is introduced into Tibet regions of China for the first time.

Peste des petits ruminants virus belongs to the genus Paramyxoviridae and the genus morbillivirus. Has similar physical, chemical and immunological properties with Rinderpest virus. Viruses are polymorphic and are generally roughly spherical in shape. The virus particles are larger than Rinderpest virus, and the nucleocapsid is a spiral hollow rod-shaped and characteristic subunit and is provided with an envelope. The virus can proliferate on testis cells and Vero cells of fetal sheep kidney, fetal sheep and newborn sheep, and produce cytopathic effect (CPE) to form syncytia.

The disease mainly infects small ruminants such as goats, sheep, white tailed deer and the like, and is prevalent in western, middle and parts of asia of africa. In epidemic areas, the disease occurs sporadically, and epidemic can occur when susceptible animals increase. The disease is mainly infected through direct contact, secretion and excrement of sick livestock are infection sources, and sick sheep in a subclinical type are particularly dangerous. The incubation period of Peste des petits ruminants is 4-5 days, and the longest period is 21 days. The natural onset is seen only in goats and sheep. The goat has serious disease, and the sheep occasionally has serious disease. The lips of some recovered goats developed aphthous lesions. The clinical symptoms of the infected animals are similar to those of cattle plague. The acute body temperature can rise to 41 ℃ and last for 3 to 5 days. The infected animals have dysphoria, dull hair, dry mouth and nose, and anorexia. Mucus flows through purulent rhinorrhea and smelly gas is exhaled. In the first 4 days of fever, the oral mucosa becomes engorged with blood, the buccal mucosa undergoes extensive damage, causing salivation, and then necrotic lesions appear, and the oral mucosa begins to appear as small rough red superficial necrotic lesions, and later turns pink, and the infected site includes lower lip, lower gum, and the like. In severe cases, necrotic lesions spread to the teeth pad, palate, cheek and its papilla, tongue, etc. In the later period, watery diarrhea with blood, severe dehydration, emaciation and body temperature reduction follow. Cough and abnormal breathing occur. The incidence rate is up to 100%, the mortality rate is 100% in severe outbreaks, and the mortality rate is not more than 50% in mild outbreaks. The serious morbidity and mortality of the young animals are high, and the disease is a disease defined in China.

The virus has special affinity to gastrointestinal tract lymphocyte and epithelial cell, so that it can cause characteristic pathological changes. Eosinophilic cytoplasmic inclusion bodies and multinucleated giant cells are typically present in infected cells. In lymphoid tissues, peste des petits ruminants virus can cause lymphocyte necrosis. Spleen, tonsil, and lymph node cells were destroyed. Multinucleated giant cells containing eosinophilic cytoplasmic inclusion bodies appeared with few intranuclear inclusion bodies. In the digestive system, the virus causes necrosis of epithelial cells deep in the markia layer, infected cells generate nuclear compaction and nuclear rupture, and multinucleated giant cells containing eosinophilic plasma inclusion bodies are formed in the epidermal germinal layer.

At present, no effective method for treating peste des petits ruminants exists, and control can be performed only by adopting methods of vaccination, killing after epidemic situations occur and regular serum monitoring. Therefore, serological diagnosis of the disease and evaluation and monitoring of vaccine immune effect are important.

The peste des petits ruminants virus antibody detection methods recommended by the world animal health organization mainly include Virus Neutralization Test (VNT) and enzyme-linked immunoassay (ELISA). The detection result of the virus neutralization test method is accurate and is a gold standard for detecting the peste des petits ruminants virus, but the method has long detection time and is not suitable for detecting a large number of samples. In contrast, enzyme-linked immunoassay has high specificity and sensitivity, has a shorter detection time than virus neutralization tests, and is suitable for detecting a large number of samples, and therefore, is widely applied to the detection of peste des petits ruminants virus antibodies.

The enzyme-linked immunoassay method for detecting the peste des petits ruminants virus antibody mainly comprises competitive enzyme-linked immunoassay (c-ELISA), blocking enzyme-linked immunoassay (b-ELISA) and indirect enzyme-linked immunoassay (indirect ELISA). The c-ELISA and the b-ELISA have higher specificity and sensitivity, and are generally accepted methods for detecting peste des petits ruminants virus antibodies in clinic, for example, the c-ELISA method is adopted by a peste des petits ruminants diagnostic kit of a French BIRAD laboratory which is commonly used internationally. However, both c-ELISA and b-ELISA need monoclonal antibody, which results in greatly increased detection cost; furthermore, c-ELISA and b-ELISA are cumbersome to operate, long in detection time (although already shortened relative to VNT), and complex in criteria. On the other hand, the conventional indirect ELISA detects antibodies in serum using intact proteins (e.g., H protein, N protein, F protein, etc.) or recombinant proteins derived from Peste des petits ruminants virus as a coating antigen at a cost lower than that of c-ELISA and b-ELISA; however, since this method uses the intact protein as an antigen, erroneous recognition and non-specific recognition of the antibody easily occur, and thus, its specificity and sensitivity are inferior to those of c-ELISA and b-ELISA.

Therefore, it is necessary to develop a peste des petits ruminants virus antibody detection kit with low detection cost, short detection time, simple operation, and high specificity and sensitivity.

Disclosure of Invention

In view of the problems in the prior art, the present invention aims to provide a peste des petits ruminants virus antibody detection kit with low detection cost, short detection time, simple operation, high specificity and sensitivity, and a polypeptide or a polypeptide combination capable of being used for preparing the kit.

The indirect ELISA method based on epitope polypeptide uses epitope polypeptide (single polypeptide of about 20 amino acids usually only contains one epitope) as coating antigen. The inventor finds that: the sensitivity of the method is low, and the sensitivity of a single polypeptide generally does not exceed 50%; the polypeptide with high sensitivity also has high false positive rate, namely low specificity. If the epitope polypeptide with high sensitivity and specificity can be found, the defects of the traditional indirect ELISA can be overcome, and a peste des petits ruminants virus antibody detection kit with the specificity and the sensitivity comparable to those of c-ELISA and b-ELISA and even higher is developed.

The inventors have conducted intensive studies to solve the above-mentioned technical problems and found, as a result, that: when using SEQ ID NO:1, SEQ ID NO:6 and the polypeptide of SEQ ID NO:7, when the polypeptide combination shown in the specification is used for detecting the peste des petits ruminants virus antibody, the sensitivity is 89%, and the specificity is 89.9%; completely comparable to the c-ELISA method and the b-ELISA method.

1. A peste des petits ruminants virus antibody (IgG) detection kit comprising one or more solid supports, and the following polypeptide combinations 6 independently linked to the one or more solid supports;

polypeptide combination 6

SEQ ID NO:1 of a polypeptide represented by the general formula (I),

the amino acid sequence of SEQ ID NO:6, and

SEQ ID NO: 7.

2. The peste des petits ruminants virus antibody (IgG) detection kit according to the above, which does not comprise other probe molecules (e.g., polypeptides, proteins, or nucleic acids).

3. Use of the polypeptide combination 6 in the preparation of a kit for detecting the presence of antibodies (IgG) against peste des petits ruminants virus in a biological sample of biological origin from a subject;

polypeptide combination 6

SEQ ID NO:1 of a polypeptide represented by the general formula (I),

SEQ ID NO:6, and

SEQ ID NO: 7.

In the present specification, the subject organism is preferably a small ruminant, more preferably a goat or sheep.

In the present specification, the biological sample may be whole blood, plasma or serum.

The polypeptide and the kit can be used for detecting whether an antibody (IgG) resisting the peste des petits ruminants virus exists in a biological sample of a biological source of a subject. In general, antibodies against peste des petits ruminants virus in a biological sample are produced by infection of a subject organism with peste des petits ruminants virus or immunization with peste des petits ruminants vaccine, and thus the polypeptide and the kit can be used to diagnose whether a subject organism is infected with peste des petits ruminants virus or immunized with peste des petits ruminants vaccine.

Detailed description of the invention

In the present specification, sensitivity means: among positive samples confirmed by the "gold standard" method, the proportion of positive samples determined by other methods was positive. The specificity refers to: among negative samples confirmed by the "gold standard" method, the proportion of negative samples was determined by other methods. For the detection of peste des petits ruminants virus antibodies, the "gold standard" in the art is the virus neutralization assay (VNT).

Polypeptide combination 6

SEQ ID NO:1 of a polypeptide represented by the general formula (I),

SEQ ID NO:6, and

SEQ ID NO: 7.

The polypeptide combination is used for detecting whether an antibody resisting the peste des petits ruminants virus exists in a biological sample of a biological source of a subject, the sensitivity and the specificity are both more than 85 percent (and are verified by a VNT method and not verified by a control kit method), and the c-ELISA method or the b-ELISA method can be compared favorably.

The polypeptide combination can be used as a detection probe for preparing a kit for detecting whether the anti-peste des petits ruminants virus antibody exists in a biological sample of a biological source of a subject.

Accordingly, the present invention also provides a peste des petits ruminants virus antibody detection kit comprising one or more solid supports, and one or more of the above combinations of polypeptides independently linked to the one or more solid supports.

In the present specification, the solid support may be one or a plurality of solid supports, but preferably one, that is, all the polypeptides are independently attached to the same solid support. In the present invention, the solid carrier is not particularly limited as long as it is a carrier which is a solid or an insoluble material. The polypeptide can be linked to the solid support by methods known to those skilled in the art.

In the present specification, the subject organism is preferably a small ruminant, more preferably a goat or sheep.

In the present specification, the biological sample may be whole blood, plasma, or serum.

In the case of detecting whether an antibody against peste des petits ruminants virus exists in a biological sample of a subject biological origin using the above-mentioned kit, when any one or more of the polypeptides in the combination of polypeptides responds to the biological sample of the subject biological origin, it is determined that an antibody against peste des petits ruminants virus exists in the biological sample of the subject biological origin (i.e., positive); conversely, when none of the polypeptides in the combination of polypeptides is responsive to a biological sample of biological origin of the subject, it is determined that antibodies against peste des petits ruminants virus are not present in the biological sample of biological origin of the subject (i.e., negative).

In the present specification, "response" means: a signal-to-noise ratio (SNR) of greater than or equal to 2, wherein signal-to-noise ratio = (polypeptide dot signal value-negative control dot signal value)/negative control dot signal value.

Examples

1. Preparation and validation of Polypeptides

The amino acid sequence of SEQ ID NO: 1. the polypeptides of 6 and 7 were synthesized by gill biochemical (shanghai) ltd and confirmed by mass spectrometry. Wherein the content of the first and second substances,

SEQ ID NO：1：SAEALFRLQAMAKILEDQEE。

SEQ ID NO：6：NQDPDKLLTVIASDKCPVVE。

SEQ ID NO：7：TPGRRTRRFVGAVLAGVALG。

2. preparation of kits (detection chips) 1-3Kit 7

The above-mentioned SEQ ID NO: 1. 6 and 7, additionally spotting a goat IgG as a positive quality control point and a PB point as a negative quality control point to prepare a detection chip, namely the kit 7.

3. Detection with a kit

Inspection step

(1) Then, 20 Xconcentrated wash (TBST: 0.4M Tris-HCl,2.74M NaCl, 2% Tween20, pH 7.2. + -. 0.2) was diluted with purified water at 1. To completely wet the surface of the test chip, about 200. Mu.L of the cleaning solution was applied to the surface of the test chip using a pipette and the test chip was immersed for a certain period of time.

(2) The serum samples to be tested were diluted with a sample diluent (0.05MPBS, 1% BSA,0.2% PVP,0.5% Tween20, pH 7.2. + -. 0.2) in accordance with a 1.

(3) Then, 200. Mu.L of the diluted serum sample was aspirated and added to the detection chip with the surface completely wet, from the detection chip from which the washing solution was discarded.

(4) The detection chip was incubated at 150 rpm for 30 minutes in a constant temperature shaker at room temperature.

(5) And discarding the serum sample, and cleaning the surface of the detection chip by using a cleaning solution.

(6) After washing, 200. Mu.L of enzyme-labeled antibody solution (rabbit anti-goat IgG-HRP, sigma, A5420) was added to the detection chip, and the detection chip was incubated at 150 rpm for 30 minutes in a constant temperature shaker at room temperature.

(7) And discarding the enzyme-labeled antibody solution, and cleaning the surface of the detection chip by using a cleaning solution.

(8) After the completion of the washing, 20. Mu.L of a luminescent substrate solution (Thermo, prod # 37074) was uniformly spread on the surface of the detection chip.

(9) And carrying out chemiluminescence imaging on the detection chip by using a gel imager, and judging the result.

(10) And judging the result: for each serum, each polypeptide in the kit was counted for response (i.e., signal-to-noise ratio (SNR) of 2 or more) and judged. That is to say that the first and second electrodes,

for the above kit 7, when SEQ ID NO:1 and/or 6 and/or 7, and determining that the peste des petits ruminants virus antibody is positive; otherwise, the result is judged to be negative.

Wherein, signal-to-noise ratio = (polypeptide dot signal value-negative control dot signal value)/negative control dot signal value. The polypeptide dot signal value refers to the chemiluminescence intensity value of the polypeptide dot read by software, and the negative control dot signal value refers to the chemiluminescence intensity value of the negative control dot read by software.

755 goat and sheep serum samples from different regions of china were respectively detected by using the kit prepared in step 2 (step 3 above) and the virus neutralization test (VNT, according to the method described in OIE (2010) peste des petits ruminants virus neutralization test), and the detection results are shown below.

TABLE 7

Sensitivity =35539/399 x 100% =89.0%

Specificity =320/356=89.9%

As can be seen from the above, the sensitivity and specificity of kit 7 were 85% or more (and were verified by VNT method, not by control kit method), which is comparable to the kits using c-ELISA method or b-ELISA method.

Sequence listing

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Claims (2)

1. A peste des petits ruminants virus antibody IgG detection kit, comprising one or more solid supports, and the following polypeptide combinations 6 independently linked to the one or more solid supports:

polypeptide combination 6

SEQ ID NO:1, SEQ ID NO:6 and the polypeptide of SEQ ID NO: 7;

the kit also comprises a goat IgG point spotted on the one or more solid carriers as a positive quality control point and a PB point as a negative quality control point.

2. Use of the following polypeptide combination 6 for the preparation of a kit for detecting the presence of anti-peste des petits ruminants virus antibody IgG in a biological sample of biological origin from a subject:

polypeptide combination 6

SEQ ID NO:1, SEQ ID NO:6 and the polypeptide of SEQ ID NO: 7;

the kit comprises one or more solid supports, and the polypeptide combination independently linked to the one or more solid supports 6;

the kit also comprises a goat IgG point spotted on the one or more solid carriers as a positive quality control point and a PB point as a negative quality control point.