Patent Publication Number: US-2005130186-A1

Title: Meningitis detection chip and fabrication method thereof and method of detecting meningitis and primer set for meningitis detection

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
      This application claims the priority benefit of Taiwan application serial no. 92135134, filed on Dec. 12, 2003.  
     BACKGROUND OF THE INVENTION  
      1. Field of the Invention  
      The present invention relates to a detection device for an illness and a fabrication method thereof, a method for detecting the illness and a primer set for detecting the illness. More particularly, the present invention relates to a meningitis detection chip and a fabrication method thereof, a method for detecting meningitis and a primer set for meningitis detection.  
      2. Description of Related Art  
      Meningitis, encephalitis, meningoencephalitis are serious illnesses of the nervous system. A variety of pathogens is responsible for these neurological infections, and there are 10 most commonly seen pathogens. An inappropriate treatment of the disease can lead to a rapid development of a life-threatening infection. A prompt and accurate diagnosis is one vital factor for treating meningitis. However, the pathogenicity and the drug resistance are not the same for the different categories or the different types of pathogens. Therefore, clinical treatment of meningitis is very difficult. For example, symptoms of viral meningitis are similar to those of other types of meningitis. However, antibiotics are not effective for treating viral meningitis. Medical practitioner normally can not provide an immediate diagnosis on the type of meningitis, for example, whether it is bacterial or viral. As a result, the patient is first hospitalized and is given antibiotics intravenously. The specific treatment is only conducted after a bacterial culture on the cerebrospinal fluid is completed, which usually takes 48 to 72 hours. In the mean time, not only undue medical expenses are incurred the intervention to the illness is delayed. Although statistically, 90% of the cases are viral meningitis, the remaining cases, which are caused by other pathogens, would complicate the clinical diagnosis of the disease.  
      The traditional methods of examinations for cerebrospinal fluid include Gram&#39;s stain, antibody based immunology test and bacterial culture. Although the Gram&#39;s stain diagnostic test is simple and fast by using a microscope, a substantial amount of the bacteria is required. The antibody based immunology test is also rapid; however, it can only test one antibody at a time. Although bacterial culture is highly sensitive, this test requires a long time to produce meaningful results. Further, the application of antibiotics may lead to a pseudo negative result in the Grams&#39; stain test or bacterial culture.  
      Microarray detection chip is one of the most important technological advancements in the high-tech field in recent years. Microarray detection chip is a high technology product that bases on a multidisciplinary effort from areas of physics, chemistry, microelectronics, precision machining and bioscience. Mircorarray detection chip can provide a large amount of probes with specific DNA sequences immobilized on a matrix, and a great deal of information is produced after the probe is reacted with a test sample (for example, DNA). Therefore, a microarray detection chip can be used for screening diseases. However, for different types of diseases, a great deal of effort must be devoted to the fabrication of the microarray detection chip in order to design a specific probe and a primer set to amplify the DNA fragment of the test sample of the patient. The current meningitis detection techniques still have not included a microarray detection chip for screening meningitis.  
     SUMMARY OF THE INVENTION  
      Accordingly, the present invention provides a specific probe designed according to the various pathogens of meningitis and a corresponding primer set, and using this microarray detection chip technique to develop an accurate and rapid meningitis detection chip and a fabrication method thereof, and a detection method.  
      In accordance to the present invention, a fabrication method for a meningitis detection chip is provided, wherein the resulting detection chip can concurrently screen a plurality of meningitis pathogens.  
      The present invention also provides a microarray detection chip for meningitis, wherein problems of an extended waiting period for a test result and an inaccurate test result are resolved.  
      The present invention further provides a detection method for meningitis for accurately and rapidly detecting whether a patient has contracted meningitis and the type of meningitis.  
      The present invention further provides a primer set for meningitis detection for amplifying a specific deoxyribonucleic acid (DNA) fragment of a patient&#39;s sample.  
      The present invention provides a fabrication method for a meningitis detection chip, wherein the method includes designing a multiple of probe sequences and these probe sequences are formed with DNA sequences assigned to SEQ ID NOs. (Sequence Identifier Number) 1 to SEQ ID No. 61. The design of the probe sequences further includes a plurality of primer sets that corresponds to the probe sequences in order to amplify specific DNA fragments of the test sample from the patient. The primer sets comprises at least 38 primer sets that are formed with the DNA sequence assigned to the SEQ ID NOs. 62 to 134. A probe synthesis step is conducted to synthesize the probes formed with DNA sequences assigned to SEQ ID NOs. 1 to SEQ ID NO. 61. Thereafter, a spotting step is performed to spot respectively these probes on a matrix.  
      The present invention provides a microarray detection chip, wherein the detection chip is applicable for detecting whether a patient has contracted meningitis. This detection chip includes immobilizing a plurality of probes on a matrix, wherein each probe is selected from the group of the DNA sequences assigned to the SEQ ID NOs. 1-61.  
      The present invention provides a detection method for meningitis, wherein this method is applicable for detecting whether a patient has meningitis. This detection method includes providing the aforementioned microarray detection chip. After obtaining a DNA sample from the patient, a plurality primer sets is used to conduct a polymerase chain reaction (PCR) to amplify a specific DNA fragment and to obtain a PCR product. The primers sets used in the PCR are selected from the 38 primer sets, which are formed with the DNA sequences assigned to the SEQ ID NO. 62-134. A hybridization procedure is then conducted for this PCR product to react with the probes on the microarray detection chip. Thereafter, the test result from the microarray detection chip is analyzed.  
      Since the microarray detection chip of the present invention employs the DNA sequence specific to meningitis, this detection chip can be used to detect whether a patient has meningitis and the type of meningitis.  
      The present invention provides a primer set for detecting meningitis, wherein this primer set is selected from one of the 38 primer sets that are formed with the DNA sequences assigned to the SEQ ID NOs. 62-134.  
      Since these primer sets (SEQ ID NOs. 62-134) are related to the specific pathogens of meningitis, using these primer sets to amplify the specific DNA fragment of the patient and the detection method, whether the patient has meningitis and the type of meningitis can be confirmed.  
      It is to be understood that both the foregoing general description and the following detailed description are exemplary, and are intended to provide further explanation of the invention as claimed. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.  
       FIG. 1  is a flow diagram illustrating the fabrication for a meningitis detection chip according to one embodiment of the invention.  
       FIG. 2  is a flow diagram illustrating the method of detecting meningitis according to one embodiment of the invention. 
    
    
     DESCRIPTION OF THE EMBODIMENTS  
      The important aspects in the fabrication of a microarray detection chip are the designs of the probes and the primers set for amplifying the DNA segments of a patient&#39;s sample. The microarray detection chip can provide accurate information only with the design of specific probes and specific primer sets. The fabrication of the microarray detection chip for meningitis and the related detection method of the present invention are based on the aforementioned concepts. Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.  
       FIG. 1  is a flow diagram illustrating the fabrication method of a meningitis detection chip according to one embodiment of the invention.  
      Referring to  FIG. 1 , a plurality of probe sequences is designed specific to the various pathogens of meningitis, wherein these probe sequences include at least the deoxyribonucleotide sequences assigned to SEQ ID NOs. (Sequence Identification Number) 1-61 (step 100). These probe sequences, for example, are formed with 15 to 30 deoxyribonucleic acids. These probe sequences are designed according to the available gene sequences of the pathogens of meningitis obtained from a gene bank.  
      It is worth to note that the typical pathogens of meningitis include virus, bacteria, fungi and protozoa. Table 1 lists the pathogens of meningitis and the corresponding SEQ ID NOs. of the probe sequences.  
                   TABLE 1                           SEQ ID NO. of the probe       Pathogens   sequences                                            Virus   hhv1   1           hhv2   2           hhv3   3           hhv4   4           hhv5   5           hhv6   6           hhv7   7           coxsackievirus a9   8           coxsackievirus b4   9           coxsackievirus b5   10           enterovirus71   11           echovirus22   12           poliovirus   13           la cross   14           St. louis   15           venezuelan equine   16           west nile   17           western equine (western)   18           western equine (eastern)   19           western equine (venezuelan)   20           Japanese B   21           central european   22           russian   23           Powassan   24           Dengue   25           Mumps   26           Measles   27           Adenovirus   28           LCMV   29           LCMV′   30           HIV-1   31           HIV-2   32           inluenza-a   33           inluenza-b   34           htlv-1   35           htlv-2   36           togavirus   37             Rickettsia typhi     38           Rabies virus   39           Parvovirus B19   40       Bacteria     Tropheryma whippelii     41             Borrelia afzelii     42             Bartonella henselae     43             Brucella abortus {grave over ( )} Brucella     44             canis {grave over ( )} Brucella melitensis {grave over ( )}               Brucella suis               Borrelia burgdorferi     45             Escherichia coli     46             Borrelia garinii     47             Streptococcus pneumoniae  R6   48             Haemophilus influenzae  Rd   49             Leptospira interrogans     50             Bacillus subtilis     51             Streptococcus milleri     52             Mycobacterium tuberculosis     53             Mycoplasma  PG50   54             Neisseria meningitidis     55             Staphylococcus aureus     56             Streptococcus pneumoniae     57       Fungi     Cryptococcus     58       Protozoa     Acanthamoeba  sp   59             Naegleria jamiesoni     60             Toxoplasma gondii     61                  
 
      The probe sequences of the SEQ ID NOs. 29 and 30 are the basis for discriminating the LCMV and the LCMV′ pathogens. Further, the probe sequence of the SEQ ID NO. 44 is basis for detecting the  Brucella abortus , the  Brucella canis , the  Brucella melitensis  and the  Brucella suis  pathogens.  
      During the step of designing the probe sequence (step 100), the design of the primer sets that correspond to these probe sequences, which are used to amplify the specific DNA sequence of the patient&#39;s sample, are also conducted. These primer sets include at least 38 primer sets, which are formed with the DNA sequences assigned to the SEQ ID NOs. 62-134. Similarly, the design of these primer sets is reliably based on the gene sequences of the pathogens of meningitis obtained from a gene bank.  
      Each primer set includes a 5′ to 3′ forward primer and a 3′ to 5′ reverse primer for amplifying the specific DNA fragments of the pathogens of meningitis. The following Table 2 lists the meningitis pathogens and the SEQ ID NOs. of the corresponding primer sets.  
                   TABLE 2                              SEQ ID NO.           of the Prime Set                             Forward   Reverse       Pathogens   Primer   Primer                                     Virus   hhv1 {grave over ( )} hhv2   62   63           hhv3   64   65           hhv4   66   67           hhv5   68   69           hhv6 {grave over ( )} hhv7   70   71           coxsackievirus a9 {grave over ( )}   72 {grave over ( )} 73   74           coxsackievirus b4 {grave over ( )}           coxsackievirus b5 {grave over ( )} enterovirus71           echovirus22   75   76           Poliovirus   77   78           la cross   79   80           St. louis   81   82           venezuelan equine   83   84           west nile   85   86           western equine {grave over ( )} eastern equine {grave over ( )}   87 {grave over ( )} 88   89           venezuelan equine           Japanese B   90   91           central european   92   93           russian   94   95           Powassan   96   97           Dengue   98   99           Mumps   100   101           Measles   102   103           Adenovirus   104   105           LCMV   106   117           HIV-1   108   109           hiv-2   110   111           inluenza-a   112   113           inluenza-b   114   115           htlv-1   116   117           htlv-2   118   119           Togavirus   120   121             Rickettsia typhi     122   123           Rabies virus   124   125           Parvovirus B19   126   127       Fungi     Cryptococcus     128   129       Bacteria       130   131       Protozoa       132 {grave over ( )} 133   134                  
 
      The DNA sequences of the SEQ ID NOs. 72-74 constitute two primer sets. In other words, the forward primer of the SEQ ID NOs. 72 and the reverse primer of the SEQ ID NOs. 73 constitute a primer set, while the forward primer of the SEQ ID NOs. 73 and the reverse primer of the SEQ ID NOs. 74 constitute another primer set. Further, these two primer sets are used to amplify the specific DNA fragments of the pathogens coxsackievirus a9, coxsackievirus b4, coxsackievirus b5 and enterovirus 71 in the test sample. Similarly, the DNA sequences of SEQ ID NOs. 87 to 89 also constitute two primer sets, wherein these primer sets are used to amplify the specific DNA fragments of the pathogens westerm equine, eastern equine, Venezuelan equine. Further, the primer sets that are formed with the DNA sequences of the SEQ ID NOs. 132 to 134 are used to amplify the specific DNA fragments of protozoa in the sample.  
      It is also worth noting that the above primer sets are not limited to be used in the detection chip of the instant invention. These primer sets, besides being used for amplifying the specific DNA fragments of a patient&#39;s sample in a detection chip, these primer sets can view as one type of detection kit. As the detection kit is being used, after the specific DNA fragment of the patient&#39;s sample is amplified, the product can be placed in an appropriate detection apparatus (not limited to the detection chip), and analysis is conducted to obtain a test result.  
      During the synthesis of the probes, the probes with the DNA sequences as assigned to SEQ ID NOs. 1-61 are formed (step 102). Further, during the synthetic step (step 102), the 5′ ends of the DNA sequences assigned to the SEQ ID NOs. 1-61 are also modified. Consequently, these probes can covalently bind with the functional groups on the matrix surface to be immobilized on the matrix surface. The modification includes a 5′ amino modification.  
      Thereafter, the synthesized probes are dissolved in deionized water to form a solution with a concentration of, for example, 200 mmole/L.  
      A spotting procedure (step 106) is then conducted to spot the probe solutions on the matrix. Depending on the number of spots required, the radius of the spots is about of 50 to 100 microns. Further, depending on the situation, each type of probe solutions can be spotted more than once. The surface area of the matrix is sufficiently large to accommodate tens to thousands of spots. If the matrix is glass, the probe solution is spotted on the matrix and is mobilized on the matrix through covalent bonding due to the 5′ amino modification to the DNA sequences in the previous probe synthesis procedure (step 102).  
      Thereafter, an incubation step is conducted to maintain the matrix in a moist and humid environment (step 108), wherein the incubation process is conducted at 37 degrees Celsius for three days continuously.  
      An oven-drying step is performed to oven-dry the matrix (step 110), wherein this oven-drying step is conducted at 80 degrees Celsius for 2 hours.  
      A matrix cleaning step is then conducted to clean the matrix (step 112), wherein the matrix cleaning step includes performing a cleaning process and a drying process. The cleaning solution used in the cleaning process is formed with a probe buffer solution and deionized water. The probe buffer solution is formed with 1×SSC and 0.1% of sodium dodecyl sulfate (SDS), while SSC is a solution with a pH of about 7 and is formed with 3M of NaCl and 0.3M of sodium citrate. The drying process is accomplished by blowing drying the matrix using nitrogen gas.  
      A blocking step is then conducted using a blocking solution to block the matrix surface that has not been blocked (step 114). The blocking solution used is, for example, a pH 7 solution formed with 1% bovine serum albumin (BSA) and 0.01 mol/L of phosphate buffer (PB).  
      A matrix cleaning step (step 116) is again conducted to clean the matrix. This matrix cleaning step includes performing a cleaning process, followed by a drying process. Further, this matrix cleaning step can be repeated for several times until the matrix is completely cleaned. The cleaning solution used in this cleaning step includes, for example, using deionized water to clean the excess blocking solution. The drying process is, for example, using nitrogen gas to blow dry the matrix. In one preferred embodiment, the matrix cleaning step is repeated, for example, for three times.  
      The fabrication of the detection chip is completed with the aforementioned method. The detection chip comprises a plurality of DNA sequences specific to meningitis. Therefore, the various pathogens of meningitis can be detected concurrently.  
      It is also worth noting that during the design of the probes (step 100), a plurality of quality control probe sequences can also design. These quality control probes are immobilized on the matrix after the synthesis step and the spotting step, etc. are performed (steps 102 to 116). The quality control probes are related to the sequence of a specific substance in the test sample, which are used to ensure the sample extracted is an effective sample to prevent misjudgment of the test result.  
      Further, using the above method, the detection chip obtained includes a plurality of probes immobilized on the matrix. Further, each probe is selected from the group of the DNA sequences assigned to the SEQ ID NOs. 1-61. Further, each probe is formed with 15 to 30 deoxyribonucleic acids. The material of the matrix is, for example, glass.  
      In another embodiment, each DNA sequence assigned to the SEQ ID NOs. 1-61 is immobilized on the matrix, wherein these DNA sequences are used to detect the pathogens of meningitis. Further, the matrix is not only disposed with these 61 probes. Depending on the situation required, the sequences recited in SEQ ID NOs. 1-61 can be repeated for several times to constitute a microarray detection chip with tens or several thousands probes.  
      Since these probes are related to the various pathogens of meningitis, they can be used to determine whether the patient has contracted meningitis and the type of meningitis.  
      The method for detecting whether a patient has contracted meningitis using the microarray detection chip, fabricated according to the above method, is detailed in the following.  
       FIG. 2  is a flow diagram illustrating a method for detecting meningitis according to one embodiment of the present invention.  
      Referring to  FIG. 2 , a microarray detection chip is provided (step 200), wherein this microarray detection chip is fabricated using, for example, the aforementioned method. Further, this microarray detection chip includes a plurality of probes specific for detecting the pathogens of meningitis. In one embodiment of the invention, besides these specific probes for detecting the pathogens of meningitis, this microarray detection chip further includes quality control probes immobilized thereon.  
      Thereafter, the sample from a patient is treated to extract the DNA from the sample (step 202), wherein the sample from the patient is, for example, cerebrospinal fluid. The cerebrospinal fluid is treated by placing in a 100 degrees Celsius water bath for about 10 minutes, followed by cooling to 0 degrees Celsius. After spinning at 13000G for 5 minutes, the supernatant is collected and is digested using lysozyme. Chloroform extraction is then conducted on the clear supernatant, followed by precipitating with ethanol to extract the DNA. If the DNA needs to be stored for a longer period time, it can be preserved at −20 degrees Celsius.  
      A PCR amplification is then conducted on the sample using a plurality of primer sets to amplify specific segments of the DNA to obtain the corresponding PCR product (step 204). The primer sets used in the PCR amplification is selected from the 38 primer sets that are formed with the DNA sequences assigned to the SEQ ID NOs. 62-134.  
      The reagent used in the PCR amplification includes at least the DNA from the sample, DNA polymerase, the above primer sets and deoxyribonucleoside triphsphate (dNTP), wherein the DNA polymerase is, for example, Tag enzyme. Further, the PCR product is labeled with a PCR product label. To label the PCR product includes, for example, using a labeled primer set, a labeled deoxyuridine triphsphate (dUTP) or a labeled dNTP and the above reagent to perform the PCR amplification. These labels are, for example, Cys, Cy5 or other appropriate fluorescent materials. The PCR product label serves as a reference in the subsequent analysis process for determining whether the PCR product has reacted with the probe.  
      In the above PCR procedure, symmetric or asymmetric multiplex polymerase chain reaction is conducted. In other words, the numbers of the forward primers and the reverse primers can be different to produce DNA with a single stranded structure in the PCR product. The single stranded DNA is subsequently hybridized with the single stranded probe on the microarray detection chip. If the numbers of the forward primer sets and the reverse primer sets are the same, a thermal denaturation procedure is conducted before the hybridization process to separate the two complementary single DNA strands before proceeding with the hybridization procedure.  
      The conditions for PCR amplification are shown as follow in Table 3.  
                                           Reaction   Duration       STEP   Temperature   Period (min.)                                            1   94   5       2   94   0.5       3   56   0.5       4   72   1       5   72   5                  
 
      In this embodiment, the PCR amplification is conducted according to the conditions in step 1, followed by repeating the conditions in steps 2 to 4 for 30 cycles, and is concluded according to the conditions in step 5.  
      Thereafter, a hybridization procedure is conducted to react the PCR product with the probes on microarray detection chip (step 206). The hybridization reaction is conducted, for example, in an environment of about 60 degrees Celsius for about 2 hours. Further, the hybridization reaction includes, for example, using a hybridization buffer, wherein the amount of the hybridization buffer used is same as the amount of the PCR product. The hybridization buffer is formed with 10×SSC and 0.1% SDS. In this procedure, if the sequence of the PCR product with the single stranded structure is complementary to the sequence of the probe, the PCR product is hybridized with the probe. Further, since the PCR product comprises a label, the type of probe that is hybridized can be detected according to this label in a subsequent process.  
      A plurality of cleaning steps is conducted to clean the microarray detection chip (step 208), wherein the cleaning solution used in these cleaning steps is, for example deionized water. In one preferred embodiment, the microarray detection chip is repeatedly cleaned for three times. Further, with these cleaning procedures (step 208), the PCR product that has not been hybridized with the probe is washed off, leaving only the PCR product that is complementary to the probe.  
      Thereafter, a result analysis step is performed on the microarray detection chip (step 210). The result analysis step includes performing a scanning procedure and a data analysis procedure, wherein the scanning procedure includes, for example, using a scanner to scan the mircroarray detection chip to obtain information on various test results. The scanner used is provided by, for example, Genomic Solutions. Further, the data analysis procedure includes, for example, using an analysis software that is compatible with the scanner to output the analysis results of the microarray detection chip. Since the PCR product after hybridization is labeled, the scanner can identify whether a label is present at the position of each probe (for example, an emission of a fluorescent signal) during the scanning step with the scanner. Therefore, based on the data analysis procedure, the type of meningitis contracted by the patient is identified. If the microarray detection chip does not include any label, it is an indication that the patient has meningitis.  
      In accordance to the present invention, the fabrication of a meningitis detection chip is provided, wherein this detection chip includes a plurality of deoxyribonucleotide sequences specific to the various types of meningitis. Therefore, the detection chip is applicable to determine whether the patient has contracted meningitis and the type of meningitis.  
      The present invention also makes use of the microarray detection chip to attain a large amount and accurate analysis results.  
      Further, these primer sets (SEQ ID NOs. 62-134) designed according to the present invention are specific to the pathogens of meningitis. Therefore, by using these primer sets to amplify the specific DNA fragments of the patient&#39;s sample and the subsequent testing method, whether the patient has meningitis and the type of meningitis are identified.  
      It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present invention without departing from the scope or spirit of the invention. In view of the foregoing, it is intended that the present invention cover modifications and variations of this invention provided they fall within the scope of the following claims and their equivalents.