Method for rapidly detecting bacterial nucleic acid in eye liquid

Abstract

The invention relates to the technical field of microbiological detection, in particular to a method for rapidly detecting nucleic acid of intraocular fluid or tear bacteria. On the basis of a constant-temperature nucleic acid amplification technology, multiple groups of amplification primers required by amplification of different pathogenic bacteria are designed, and detection primers with high detection sensitivity, strong specificity and good stability are obtained through screening; secondly, designing and optimizing efficient pathogen cracking conditions and preparation of related reagents according to biological characteristics of target pathogens; the primer group and the reagent obtained through optimization are used for detecting pathogenic bacteria, the sample size demand is low, the sensitivity and the specificity are high, and an effective technical means is provided for rapid detection of bacterial infection on site (outpatient service, ward or operating room) after cataract operation.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for rapid detection of bacterial nucleic acid in eye fluid. Background Art

[0002] Age-related cataract (ARC) is the leading reversible blinding eye disease worldwide, accounting for half of all cases of vision loss caused by various reasons. There is still a lack of effective therapeutic drugs, and surgery is currently the only effective treatment. Infectious endophthalmitis is a serious complication of cataract surgery. Most bacteria, especially Staphylococcus epidermidis, exist in normal periocular tissues such as the conjunctival sac and eyelid eyelashes, as well as in the air. These normal ocular flora or other exogenous bacteria enter the eye through surgical instruments, perfusion fluid, and artificial lenses during cataract surgery, grow under appropriate conditions, and cause intraocular infection. However, it should be noted that if infectious endophthalmitis occurs, it is easy to affect the treatment effect, and in severe cases it can cause permanent loss of vision. Therefore, early prevention of intraocular pathogens and effective treatment of post-cataract surgery infection are of great clinical significance.

[0003] At present, the common pathogens of postoperative infectious endophthalmitis in large and medium-sized ophthalmology institutions in my country include coagulase-negative surface staphylococci, Staphylococcus aureus, Streptococcus spp., and Gram-negative bacilli such as Pseudomonas aeruginosa. Other common bacteria include hemolytic streptococci, Streptococcus pneumoniae, and Bacillus cereus. The traditional clinical bacterial culture method has a long diagnosis cycle of 2 to 7 days, high sample storage conditions, and a low positive rate. The polymerase chain reaction (PCR) takes a long time, the amplified product needs to be confirmed by electrophoresis, the professional equipment is expensive, and it is easy to be contaminated and has a high false positive rate, which limits its application. DNA microarray analysis: It is expensive and the sample processing is complicated, so it cannot be widely used in the detection of pathogens. Next-generation sequencing technology (NGS and mNGS): This technology faces a series of challenges in practical application, such as cost, technology, and data issues. Immunological detection methods may have cross-reactions, matrix effects, and interference, resulting in low specificity or sensitivity or false negative results. Current diagnostic methods for identifying bacterial pathogens in aqueous humor are time-consuming and often fail to provide timely results, thus delaying the initiation of appropriate antimicrobial therapy. The need for a new rapid test for ocular fluid bacteria and a fast and accurate test are critical to improving clinical outcomes in patients at high risk for postoperative endophthalmitis. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for rapid detection of bacterial nucleic acids in ocular fluid.

[0005] The present invention provides a primer set for detecting bacteria in ocular fluid, which includes at least one of a primer set for detecting Staphylococcus epidermidis, a primer set for detecting Pseudomonas aeruginosa, a primer set for detecting Staphylococcus aureus, a primer set for detecting hemolytic Streptococcus, and / or a primer set for detecting Streptococcus pneumoniae;

[0006] The detection primer set of Staphylococcus epidermidis includes:

[0007] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33 or SEQ ID NO: 37;

[0008] and a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34 or SEQ ID NO:38;

[0009] and an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35 or SEQ ID NO:39; and

[0010] A downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36 or SEQ ID NO:40;

[0011] The detection primer set of Pseudomonas aeruginosa comprises:

[0012] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:58 or SEQ ID NO:62; and

[0013] a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:59 or SEQ ID NO:63; and

[0014] An upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:60 or SEQ ID NO:64; and

[0015] A downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:61 or SEQ ID NO:65; and

[0016] An upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57 or SEQ ID NO:66; and / or

[0017] A downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO:46 or SEQ ID NO:52;

[0018] The detection primer set of Staphylococcus aureus comprises:

[0019] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:80 or SEQ ID NO:84; and

[0020] a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:81 or SEQ ID NO:85; and

[0021] An upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:82 or SEQ ID NO:86; and

[0022] The nucleotide sequence is shown in SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:83 or SEQ ID NO:87; downstream internal primer (BIP);

[0023] A downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO:75;

[0024] The detection primer set of the hemolytic streptococcus comprises:

[0025] An upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:100 or SEQ ID NO:104; and

[0026] a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:89, SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:101 or SEQ ID NO:105; and

[0027] An upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102 or SEQ ID NO:106; and

[0028] The nucleotide sequence is shown in SEQ ID NO:91, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:103 or SEQ ID NO:107; downstream internal primer (BIP);

[0029] The detection primer set of Streptococcus pneumoniae includes:

[0030] An upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 108, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO: 122 or SEQ ID NO: 126; and

[0031] A downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123 or SEQ ID NO: 127; and

[0032] An upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124 or SEQ ID NO: 128; and

[0033] A downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 111, SEQ ID NO: 117, SEQ ID NO: 121, SEQ ID NO: 125 or SEQ ID NO: 129; and

[0034] An upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO: 112 or SEQ ID NO: 130; and / or

[0035] A downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO: 113 or SEQ ID NO: 131;

[0036] The detection primer set of the Bacillus cereus comprises:

[0037] The upstream outer primer (F3) with a nucleotide sequence as shown in SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144 or SEQ ID NO: 148, and

[0038] A downstream external primer (B3) with a nucleotide sequence as shown in SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145 or SEQ ID NO: 149, and

[0039] The upstream internal primer (FIP) with a nucleotide sequence as shown in SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146 or SEQ ID NO: 150, and

[0040] The nucleotide sequence is shown as SEQ ID NO:135, SEQ ID NO:139, SEQ ID NO:143, SEQ ID NO:147 or SEQ ID NO:151 as the downstream internal primer (BIP).

[0041] Furthermore, in the primer set of the present invention, the detection primer set for Staphylococcus epidermidis includes:

[0042] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 2, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 3, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 4; and / or

[0043] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:5, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:6, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:7, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:8; and / or

[0044] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:9, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:10, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:11, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:12; and / or

[0045] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 13, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 14, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 15, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 16; and / or

[0046] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 17, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 18, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 19, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 20; and / or

[0047] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 21, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 22, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 23, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 24; and / or

[0048] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 25, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 26, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 27, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 28; and / or

[0049] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:29, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:30, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:31, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:32; and / or

[0050] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:33, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:34, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:35, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:36; and / or

[0051] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:37, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO:38, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:39, and a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:40;

[0052] The detection primer set of Pseudomonas aeruginosa comprises:

[0053] An upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:41, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO:42, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:43, a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:44, an upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO:45, and a downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO:46;

[0054] and / or an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:47, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO:48, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:49, a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:50, an upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO:51, and a downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO:52; and / or

[0055] An upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:53, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO:54, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:55, a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:56, and an upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO:57; and / or

[0056] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:58, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:59, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:60, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:61;

[0057] and / or an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:62, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO:63, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:64, a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:65, and an upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO:66;

[0058] The detection primer set of Staphylococcus aureus comprises:

[0059] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:67, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:68, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:69, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:70; and / or

[0060] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:71, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO:72, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:73, a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:74, and a downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO:75; and / or

[0061] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:76, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:77, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:78, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:79; and / or

[0062] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:80, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:81, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:82, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:83; and / or

[0063] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO:84, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO:85, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:86, and a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:87;

[0064] The detection primer set of the hemolytic streptococcus comprises:

[0065] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:88, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:89, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:90, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:91; and / or

[0066] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:92, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:93, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:94, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:95; and / or

[0067] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO:96, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO:97, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO:98, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO:99; and / or

[0068] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 100, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 101, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 102, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 103; and / or

[0069] An upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 104, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 105, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 106, and a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 107;

[0070] The detection primer set of Streptococcus pneumoniae includes:

[0071] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 108, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 109, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 110, a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 111, an upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO: 112, and a downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO: 113;

[0072] and / or an upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 114, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 115, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 116, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 117; and / or

[0073] An upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 118, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 119, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 120, and a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 121; and / or

[0074] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 122, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 123, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 124, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 125; and / or

[0075] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 126, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 127, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 128, a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 129, an upstream loop primer (LF) having a nucleotide sequence as shown in SEQ ID NO: 130, and a downstream loop primer (LB) having a nucleotide sequence as shown in SEQ ID NO: 131;

[0076] The detection primer set of the Bacillus cereus comprises:

[0077] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 132, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 133, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 134, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 135; and / or

[0078] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 136, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 137, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 138, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 139; and / or

[0079] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 140, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 141, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 142, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 143; and / or

[0080] An upstream external primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 144, a downstream external primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 145, an upstream internal primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 146, and a downstream internal primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 147; and / or

[0081] an upstream outer primer (F3) having a nucleotide sequence as shown in SEQ ID NO: 148, a downstream outer primer (B3) having a nucleotide sequence as shown in SEQ ID NO: 149, an upstream inner primer (FIP) having a nucleotide sequence as shown in SEQ ID NO: 150, and a downstream inner primer (BIP) having a nucleotide sequence as shown in SEQ ID NO: 151;

[0082] The concentration of each primer in the primer set is: upstream external primer (F3) 0.1 μM to 0.2 μM; downstream external primer (B3) 0.1 μM to 0.2 μM; upstream internal primer (FIP) 0.8 μM to 1.6 μM; downstream internal primer (BIP) 0.8 μM to 1.6 μM; upstream loop primer (LF) 0.4 μM to 0.8 μM; downstream loop primer (LB) 0.4 μM to 0.8 μM;

[0083] Furthermore, the concentration of each primer in the primer set is: upstream external primer (F3) 0.2 μM, downstream external primer (B3) 0.2 μM, upstream internal primer (FIP) 1.6 μM, downstream internal primer (BIP) 1.6 μM, upstream loop primer (LF) 0.8 μM, downstream loop primer (LB) 0.8 μM.

[0084] The present invention realizes the detection of six pathogens in intraocular fluid or tears; based on the constant temperature nucleic acid amplification technology, the present invention designs multiple groups of amplification primers required for amplification of different pathogens, and then obtains detection primers with high detection sensitivity, strong specificity and good stability through screening; the experimental results show that;

[0085] Among the detection primer sets for Staphylococcus epidermidis, SE-19 has the best detection effect;

[0086] Among the primer sets for detecting Pseudomonas aeruginosa, primer set 1 has the best detection effect;

[0087] Among the primer sets for detecting Staphylococcus aureus, primer set 3 has the best detection effect;

[0088] Among the primer sets for detecting hemolytic streptococci, primer set 3 has the best detection effect;

[0089] Among the primer sets for detecting Streptococcus pneumoniae, primer set 4 has the best detection effect;

[0090] Among the primer sets for detecting Bacillus cereus, primer set 2 has the best detection effect;

[0091] The present invention provides a detection reagent, which includes a detection mixture and the primer set of the present invention; the detection mixture includes: Bst DNA polymerase, dNTP, betaine, magnesium sulfate, Bst LF Buffer with Mg 2+ and / or at least one of an indicator reagent.

[0092] Furthermore, the indicator reagent is selected from SYBR Green I and / or calcein.

[0093] In the present invention, the detection mixture includes: 0.15μM to 0.4μM Bst DNA polymerase, 0.2mM to 10mM dNTP, 0M to 1.2M betaine, 2mM to 10mM magnesium sulfate, 1×Bst LF Buffer with Mg 2+ , 1-2 mM calcein;

[0094] In a specific embodiment of the present invention, the detection reagent includes: 0.4 μM Bst DNA polymerase, 10 mM dNTP, 1.2 M betaine, 10 mM magnesium sulfate, 1× Bst LF Buffer with Mg 2+ , 2 mM calcein.

[0095] The concentrations of the various substances in the test mixture described here are all working concentrations.

[0096] Among them, 1×Bst LF Buffer with Mg 2+， The magnesium ion content is 2mM, and the total magnesium ion content in the detection reagent is controlled at 4-12mM;

[0097] In the present invention, the amplification reagent ratios and pathogen lysis conditions of different pathogens are optimized. The test results show that the detection effect (such as detection limit, sensitivity, specificity, accuracy, anti-interference ability, stability, etc.) after treating the pathogens with the reagent ratios and lysis conditions described in the present invention is significantly better than other treatments and existing technologies.

[0098] The present invention provides a kit, which includes at least one of a positive quality control product and / or a negative quality control product and at least one of the following i) to ii):

[0099] i), the primer set of the present invention;

[0100] ii), the detection reagent of the present invention.

[0101] The present invention provides a method for detecting bacteria in intraocular fluid for non-diagnostic purposes, characterized in that the sample is detected using at least one of the following I) to III):

[0102] 1), the primer set of the present invention;

[0103] II), the detection reagent of the present invention;

[0104] III), the kit of the present invention.

[0105] Furthermore, the detection methods described in the present invention include detection methods for diagnostic purposes and non-diagnostic purposes, which are not limited in the present invention. The detection methods for non-diagnostic purposes include detection of environmental samples, in vitro samples, or detection for scientific research purposes, wherein the environmental samples include but are not limited to: food, drinking water, domestic water, domestic wastewater, or surface swabs of objects; in vitro samples include but are not limited to conjunctival sac secretions, tears, aqueous humor, and / or vitreous humor, wherein the in vitro samples are not directly implemented on living human or animal bodies.

[0106] Furthermore, the detection method of the present invention comprises the following steps: mixing the detection reagent according to any one of claims 3 to 5 with a sample to be tested and reacting the mixture, and judging whether the sample is infected with pathogenic bacteria according to the reaction result;

[0107] The reaction program was 63 °C, 45 min;

[0108] The criteria for the interpretation are: Ct value < 35, and the amplification curve is the standard S curve for positive, and the others are negative.

[0109] The samples include conjunctival sac secretions, tears, aqueous humor and / or vitreous humor.

[0110] The present invention provides a detection primer set, a detection reagent, a kit and a detection method for pathogens in ocular fluid after cataract surgery. Based on the constant temperature nucleic acid amplification technology, the present invention designs multiple groups of amplification primers required for amplification of different pathogens, and screens and obtains detection primers with high detection sensitivity, strong specificity and good stability; secondly, according to the biological characteristics of the target pathogen, the efficient pathogen lysis conditions and the preparation of related reagents are designed and optimized. The optimized primer set and reagents are used for the detection of pathogens, with a small sample volume requirement, high sensitivity and specificity, and provide an effective technical means for the on-site (outpatient clinic, ward or operating room) rapid detection of bacterial infection after cataract surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1A schematic diagram showing the detection effect of surface Staphylococcus aureus in intraocular fluid or tear samples;

[0112] Figure 2 A schematic diagram showing the detection effect of Pseudomonas aeruginosa in intraocular fluid or tear samples;

[0113] Figure 3 A schematic diagram showing the detection effect of Staphylococcus aureus in intraocular fluid or tear samples;

[0114] Figure 4 A schematic diagram showing the detection effect of hemolytic streptococci in intraocular fluid or tear samples;

[0115] Figure 5 A schematic diagram showing the detection effect of Streptococcus pneumoniae in intraocular fluid or tear samples;

[0116] Figure 6 Schematic diagram showing the detection effect of Bacillus cereus in intraocular fluid or tear samples. DETAILED DESCRIPTION

[0117] The present invention provides a method for rapid detection of bacterial nucleic acid in ocular fluid. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0118] The rapid detection method of the present invention detects 6 types of bacteria, including: Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Bacillus cereus. Other bacteria, fungi, viruses or parasites that may cause postoperative infection of cataract surgery can also be detected by the same method.

[0119] The present invention adopts a method for rapid detection of bacteria after cataract surgery in ophthalmology, which does not require large equipment, toxic chemical reagents, or complicated operation steps, and can be used in conjunction with LAMP technology for rapid nucleic acid amplification detection. The overall detection process from sample processing to final result interpretation only takes 45 minutes, and does not require professional personnel and professional site equipment. This allows the detection of bacteria after cataract surgery to be freed from the laboratory, and the detection can be completed quickly on site (outpatient clinic, ward or operating room), and guide the patient's subsequent timely treatment.

[0120] The loop-mediated isothermal amplification (LAMP) method is used to perform high-precision detection of the extracted trace bacterial genomic DNA. It is simple to operate, time-saving, rapid and sensitive, thus making up for the shortcomings of the existing traditional culture method. It can be used on-site (outpatient clinic, ward or operating room) for the rapid detection of bacteria infected after cataract surgery, which can provide guidance for the early prevention and treatment of invasive infections.

[0121] The specific scheme of the present invention:

[0122] The present invention provides a method for rapid detection of bacterial nucleic acid after cataract surgery in ophthalmology on-site (outpatient clinic, ward or operating room), and the collected specimens include tears, aqueous humor and vitreous humor. This method can greatly simplify the sample preparation steps and shorten the overall detection time. In addition, low-concentration microorganisms in trace samples (50-200 μl) can be detected without cultivation, thereby improving detection efficiency.

[0123] For the main ocular pathogens and common pathogens of postoperative endophthalmitis, including Staphylococcus epidermidis (ATCC26069), Pseudomonas aeruginosa (CMCC63302), Staphylococcus aureus (CICC21600), Streptococcus hemolyticus (CMCC32210), Streptococcus pneumoniae (CMCC31924) and Bacillus cereus (CMCC63302), all related pathogen gene sequences were first compared by BLAST to determine the specific gene sequence. Based on the constant temperature nucleic acid amplification technology, multiple sets of amplification primers required for different amplifications were designed, and detection primers with high detection sensitivity, strong specificity and good stability were screened. At the same time, a complete pathogen molecular database for diagnosis was established. Secondly, according to the biological characteristics of the target pathogen, we design and optimize efficient pathogen lysis conditions and the preparation of related reagents to complete the technical development and optimization that meets clinical needs. Since the intraocular fluid sample volume is extremely small, about 0.05 to 0.2 mL, high sensitivity and specificity are required.

[0124] A method for rapid extraction and detection of nucleic acids of six endophthalmitis-related bacteria in aqueous humor after cataract surgery, comprising the following steps:

[0125] 1. Extraction of genomic DNA from the sample to be tested: Take an appropriate amount of eye fluid sample directly without nucleic acid extraction, or centrifuge it in an EP tube at 12000rpm for 5min, transfer the supernatant to a new 1.5mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0126] 2. Loop-mediated isothermal amplification: Using the sample DNA to be tested as a template, the extracted bacterial genomic DNA is detected using the loop-mediated isothermal amplification (LAMP) method, the fluorescence signal is collected, and the results are analyzed according to the fluorescence amplification curve, so as to detect trace bacteria of trace endophthalmitis after cataract surgery; the bacteria include 6 bacteria related to endophthalmitis: a. Pseudomonas aeruginosa, b. Staphylococcus aureus, c. Staphylococcus aureus, d. Hemolytic Streptococcus, e. Streptococcus pneumoniae, and f. Bacillus cereus.

[0127] The steps for detecting trace bacteria in post-cataract surgery endophthalmitis are as follows:

[0128] 1. Collect eye samples: conjunctival sac secretions or tears, aqueous humor and vitreous humor.

[0129] 2. Take an appropriate amount of eye fluid sample directly without nucleic acid extraction, or centrifuge it in an EP tube at 12000rpm for 5min, transfer the supernatant to a new 1.5mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0130] 3. Take 1-5 μL of the sample DNA template to be tested and 20-25 μL of LAMP reaction solution into the PCR tube, mix well and centrifuge briefly to remove the liquid on the lid and tube wall;

[0131] 4. Place the PCR tube in a portable PCR instrument, react at 60-70°C for 45 minutes, and detect the fluorescence signal every 1 minute;

[0132] 5. Determine the test results through the amplification curve.

[0133] The LAMP reaction solution includes 8U / reaction of 0.15μM to 0.4μM Bst DNA polymerase large fragment, 0.2mM to 10mM dNTP, 0M to 1.2M betaine, 2mM to 10mM magnesium sulfate, 1× Bst LF Buffer with Mg 2+, 1×SYBRGreen I or calcein, 0.1μM~0.2μM outer primer F3, 0.1μM~0.2μM outer primer B3, 0.8μM~1.6μM inner primer FIP, 0.8μM~1.6μMμM inner primer BIP, 0.4μM~0.8μM loop-mediated primer LF, 0.4μM~0.8μM loop-mediated primer LB; the LAMP primers are designed for bacterial-specific genes, and the LAMP reaction system is optimized in real time.

[0134] The criteria for interpreting the results are as follows:

[0135] 1. Judge by gel electrophoresis: if there is a characteristic ladder-shaped band in the reaction solution after the reaction, it is a positive result, and if there is no characteristic ladder-shaped band, it is a negative result;

[0136] 2. If the Ct value is less than 35 and the amplification curve is a standard S curve, it is positive; otherwise, it is negative.

[0137] 3. If SYBR Green I (DNA binding dye) is used, after the reaction is completed, yellow-green is positive and orange is negative;

[0138] 4. If calcein (colorimetric indicator) is used, after the reaction is completed, green is positive, and no green is negative;

[0139] The advantages of the technical solution provided by the present invention are:

[0140] 1. The total time required for detection is shorter. The ocular fluid provided by the present invention can be directly amplified without nucleic acid extraction, which greatly simplifies the operation steps and time compared to nucleic acid extraction using commercial test kits. At the same time, the LAMP method provided by the present invention is shorter than the PCR method (LAMP detection is about 45 minutes, and PCR detection is about 120 minutes), and the LAMP reaction has lower requirements for sample purity. There is no need for professional laboratory doctors and laboratories, and doctors or nurses can conduct detection at the bedside or operating table. Compared with the culture method that takes 2 to 7 days to produce results, the present invention can complete the test within 45 minutes.

[0141] 2. Small sample volume required: This method only requires 50 to 200 μl of ocular samples to be directly processed.

[0142] 3. This method has strong specificity and high sensitivity. No nucleic acid extraction is required for 6 kinds of bacteria, including Staphylococcus aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Bacillus cereus. The direct amplification detection sensitivity is 10-20 copies / reaction, 1000 copies / mL. The LAMP primers are designed for the specific nucleic acid sequences of bacteria, eliminating the interference of non-target bacteria and fungi. There are no false positives in the cross-experiments between the 6 different detection targets, and there are no false positives in the cross-experiments with common pathogenic microorganisms (Klebsiella pneumoniae, Haemophilus influenzae, Catmorella, Acinetobacter baumannii, Legionella pneumophila, Listeria monocytogenes, Vibrio parahaemolyticus, Enterobacter sakazakii, Salmonella, Shigella dysenteriae, Escherichia coli O-157, Vibrio cholerae, Campylobacter jejuni).

[0143] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described in conjunction with the following examples: Example 1 Detection of surface Staphylococcus aureus in ocular fluid samples after cataract surgery

[0144] This embodiment provides a method for rapid detection of bacterial nucleic acid after cataract surgery, taking the detection of surface Staphylococcus aureus in an ocular fluid sample as an example.

[0145] 1. The primer set sequences for surface Staphylococcus aureus are shown in Table 1.

[0146] Table 1: Primer sequences for isothermal amplification of Staphylococcus epidermidis nucleic acid

[0147]

[0148]

[0149] 2. Ocular fluid processing method: After the ocular specimens (aqueous humor, vitreous body, and tears) are directly or completely lysed, the EP tube is centrifuged at 12,000 rpm for 5 min, and the supernatant is transferred to a new 1.5 mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0150] 3. 10× primer mixture preparation: Use the primers in Table 1 above to prepare 10× primer mixture, and the primer composition is as shown in Table 2 below:

[0151] Table 2. 10× Primer Mix for Detection of Staphylococci on Surfaces

[0152]

[0153] 4. The ratio of primers, LAMP reaction solution and samples to be tested in the LAMP reaction system is as shown in Table 3:

[0154] Table 3. LAMP reaction system for detecting surface Staphylococci

[0155] Components Volume (μL) 2×LAMP Master Mix (LAMP reaction solution) 10～15 10× Primer Mix 1～2 Samples to be tested, positive / negative samples 1～5 Sterile purified water 3～10 Total volume 20～25

[0156] The LAMP reaction solution can be purchased 2×LAMP Master Mix with Dye (nearshore protein; E223).

[0157] 5. Rapid extraction and detection of nucleic acid from intraocular fluid (aqueous humor, vitreous body) or tears The steps for detecting surface Staphylococci are as follows:

[0158] Specifically, the steps of using the rapid nucleic acid extraction and detection method of intraocular fluid (aqueous humor, vitreous body) or tears to detect surface staphylococci are as follows:

[0159] (1) Obtaining samples for LAMP testing in clinic;

[0160] (2) Prepare a LAMP reaction system by mixing LAMP reaction solution, 10× primer mixture, sample to be tested and sterilized purified water according to the ratio in Table 3 above;

[0161] (3) Place the prepared LAMP reaction system into a portable PCR instrument (commercially available), react at 63°C for 45 min, and collect fluorescence every 1 min;

[0162] (4) After the reaction is completed, the results are interpreted on the instrument.

[0163] The result interpretation standard is that the Ct value is less than 35 and the amplification curve is the standard S curve for positive, and the others are negative. Figure 1 As shown, this method can be used to detect surface Staphylococcus aureus in ocular fluid samples. The detection limit of surface Staphylococcus aureus can reach 10-20 copies / reaction, 1000 copies / mL, among which SE-19 is the best. The other groups could not detect and were negative.

[0164] Example 2 Detection of Pseudomonas aeruginosa in ocular fluid samples after cataract surgery

[0165] Based on the above Example 1, this example provides a method for rapid detection of bacterial nucleic acid after cataract surgery, taking detection of Pseudomonas aeruginosa in ocular fluid samples as an example. The sequence of the primer set for Pseudomonas aeruginosa is shown in Table 4.

[0166] Table 4. Pseudomonas aeruginosa primer set sequences

[0167]

[0168] Intraocular fluid sample testing method:

[0169] The first step is to centrifuge the intraocular fluid (aqueous humor, vitreous body) or tears directly or after lysis at 12000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0170] Step 2: Prepare 10× primer mixture using the primers in Table 4 above, and the primer composition is as shown in Table 2;

[0171] Step 3: Prepare a LAMP reaction system by mixing the LAMP reaction solution, 10× primer mixture, the sample to be tested and sterilized purified water according to the ratio in Table 3 above;

[0172] Step 4: Place the prepared LAMP reaction system into a portable PCR instrument (commercially available), react at 63°C for 45 minutes, and collect fluorescence every 1 minute;

[0173] Step 5: After the reaction is completed, interpret the results on the instrument.

[0174] The result interpretation standard is that the Ct value is less than 35 and the amplification curve is the standard S curve for positive, and the others are negative. Figure 2 As shown, it is proved that this method can complete the detection of Pseudomonas aeruginosa in ocular fluid samples, and the detection limit of Pseudomonas aeruginosa can reach 10-20 copies / reaction, 1000 copies / mL, among which primer set 1 is the best.

[0175] Implementation 3 Detection of Staphylococcus aureus in ocular fluid samples

[0176] Based on the above-mentioned Example 1, this example provides a method for rapid detection of bacterial nucleic acid after cataract surgery, taking the detection of Staphylococcus aureus in an ocular fluid sample as an example.

[0177] The primer sequences of the primer set used for Staphylococcus aureus detection are shown in Table 5, which were synthesized by Anhui General Biotechnology and purified by PAGE.

[0178] Table 5. Primer sequences for isothermal amplification of nucleic acids for detecting Staphylococcus aureus

[0179]

[0180] Intraocular fluid sample testing method:

[0181] The first step is to centrifuge the intraocular fluid (aqueous humor, vitreous body) or tears directly or after lysis at 12000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0182] Step 2: Prepare 10× primer mixture using the primers in Table 4 above, and the primer composition is as shown in Table 2;

[0183] Step 3: Prepare a LAMP reaction system by mixing the LAMP reaction solution, 10× primer mixture, the sample to be tested and sterilized purified water according to the ratio in Table 3 above;

[0184] Step 4: Place the prepared LAMP reaction system into a portable PCR instrument (commercially available), react at 63°C for 45 minutes, and collect fluorescence every 1 minute;

[0185] Step 9: After the reaction is completed, interpret the results on the instrument.

[0186] The result interpretation standard is that the Ct value is less than 35 and the amplification curve is the standard S curve for positive, and the others are negative. Figure 3 As shown, it is proved that this method can complete the detection of Staphylococcus aureus in ocular fluid samples, and the detection limit of Staphylococcus aureus can reach 10-20 copies / reaction, 1000 copies / mL; among them, primer set 3 is the best.

[0187] Example 4 Detection of hemolytic streptococci in ocular fluid samples after cataract surgery

[0188] Based on the above-mentioned Example 1, this example provides a method for rapid detection of bacterial nucleic acid after cataract surgery to detect hemolytic streptococci in ocular fluid samples.

[0189] The sequences of the primer sets for hemolytic streptococci are shown in Table 6;

[0190] The eye sample testing method is as follows:

[0191] The first step is to centrifuge the intraocular fluid (aqueous humor, vitreous body) or tears directly or after lysis at 12000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0192] Step 2: Prepare 10× primer mixture using the primers in Table 4 above, and the primer composition is as shown in Table 2;

[0193] Step 3: Prepare a LAMP reaction system by mixing the LAMP reaction solution, 10× primer mixture, the sample to be tested and sterilized purified water according to the ratio in Table 3 above;

[0194] Step 4: Place the prepared LAMP reaction system into a portable PCR instrument (commercially available), react at 63°C for 45 minutes, and collect fluorescence every 1 minute;

[0195] Step 5: After the reaction is completed, interpret the results on the instrument.

[0196] Table 6. Primer sequences for isothermal amplification of nucleic acid of hemolytic streptococci

[0197]

[0198] The result interpretation standard is that the Ct value is less than 35 and the amplification curve is the standard S curve for positive, and the others are negative. Figure 4 As shown, this method can be used to detect hemolytic streptococci in ocular fluid samples. The detection limit of hemolytic streptococci can reach 10-20 copies / reaction, 1000 copies / mL, among which primer set 3 is the best.

[0199] Example 5 Detection of Streptococcus pneumoniae in ocular fluid samples after cataract surgery

[0200] The method for rapid detection of bacterial nucleic acid after cataract surgery is used to detect Streptococcus pneumoniae in ocular fluid samples. The sequence of the Streptococcus pneumoniae primer set is shown in Table 7.

[0201] Table 7. Primer sequences for isothermal amplification of nucleic acid of Streptococcus pneumoniae

[0202]

[0203]

[0204] Eye sample testing method:

[0205] The first step is to centrifuge the intraocular fluid (aqueous humor, vitreous body) or tears directly or after lysis at 12000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0206] Step 2: Prepare 10× primer mixture using the primers in Table 4 above, and the primer composition is as shown in Table 2;

[0207] Step 3: Prepare a LAMP reaction system by mixing the LAMP reaction solution, 10× primer mixture, the sample to be tested and sterilized purified water according to the ratio in Table 3 above;

[0208] Step 4: Place the prepared LAMP reaction system into a portable PCR instrument (commercially available), react at 63°C for 45 minutes, and collect fluorescence every 1 minute;

[0209] Step 5: After the reaction is completed, interpret the results on the instrument.

[0210] The result interpretation standard is that the Ct value is less than 35 and the amplification curve is the standard S curve for positive, and the others are negative. Figure 5As shown, this method can be used to detect Streptococcus pneumoniae in intraocular fluid samples. The detection limit of Streptococcus pneumoniae can reach 10-20 copies / reaction, 1000 copies / mL, among which primer set 4 is the best.

[0211] Example 6 Detection of Bacillus cereus in ocular fluid samples

[0212] Based on the above Example 1, this example provides a method for rapid detection of bacterial nucleic acid after cataract surgery, taking detection of Bacillus cereus in ocular fluid samples as an example. The sequence of the Bacillus cereus primer set is shown in Table 8.

[0213] Table 8. Primer sequences for isothermal amplification of nucleic acid of Bacillus cereus

[0214]

[0215] Eye sample testing method:

[0216] The first step is to centrifuge the intraocular fluid (aqueous humor, vitreous body) or tears directly or after lysis at 12000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL sterile EP centrifuge tube to obtain the sample to be tested for LAMP.

[0217] Step 2: Prepare 10× primer mixture using the primers in Table 4 above, and the primer composition is as shown in Table 2;

[0218] Step 3: Prepare a LAMP reaction system by mixing the LAMP reaction solution, 10× primer mixture, the sample to be tested and sterilized purified water according to the ratio in Table 3 above;

[0219] Step 4: Place the prepared LAMP reaction system into a portable PCR instrument (commercially available), react at 63°C for 45 minutes, and collect fluorescence every 1 minute;

[0220] Step 5: After the reaction is completed, interpret the results on the instrument.

[0221] The result interpretation standard is that the Ct value is less than 35 and the amplification curve is the standard S curve for positive, and the others are negative. Figure 6 As shown, this method can be used to detect Bacillus cereus in ocular fluid samples. The detection limit of Bacillus cereus can reach 10-20 copies / reaction, 1000 copies / mL, among which primer set 2 is the best.

[0222] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for detecting bacteria in intraocular fluid, characterized in that: Including at least one of a detection primer set for Staphylococcus epidermidis, a detection primer set for Pseudomonas aeruginosa, a detection primer set for Staphylococcus aureus, a detection primer set for hemolytic Streptococcus, a detection primer set for Streptococcus pneumoniae and / or a detection primer set for Bacillus cereus; The detection primer set of Staphylococcus epidermidis includes: An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33 or SEQ ID NO: 37; and a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34 or SEQ ID NO:38; and an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35 or SEQ ID NO:39; and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36 or SEQ ID NO:40; The detection primer set of Pseudomonas aeruginosa comprises: An upstream external primer having a nucleotide sequence as shown in SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:58 or SEQ ID NO:62; and A downstream external primer having a nucleotide sequence as shown in SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:59 or SEQ ID NO:63; and An upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:60 or SEQ ID NO:64; and A downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:61 or SEQ ID NO:65; and An upstream loop primer having a nucleotide sequence as shown in SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57 or SEQ ID NO:66; and / or a downstream loop primer having a nucleotide sequence as shown in SEQ ID NO:46 or SEQ ID NO:52; The detection primer set of Staphylococcus aureus comprises: An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:80 or SEQ ID NO:84; and A downstream external primer having a nucleotide sequence as shown in SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:81 or SEQ ID NO:85; and An upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:82 or SEQ ID NO:86; and The nucleotide sequence is shown as SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:83 or SEQ ID NO:87; downstream internal primer; A downstream loop primer having a nucleotide sequence as shown in SEQ ID NO:75; The detection primer set of the hemolytic streptococcus comprises: An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:100 or SEQ ID NO:104; and A downstream external primer having a nucleotide sequence as shown in SEQ ID NO:89, SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:101 or SEQ ID NO:105; and An upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102 or SEQ ID NO:106; and The nucleotide sequence is shown as SEQ ID NO:91, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:103 or SEQ ID NO:107; downstream internal primer; The detection primer set of Streptococcus pneumoniae includes: An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO: 108, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO: 122 or SEQ ID NO: 126; and A downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123 or SEQ ID NO: 127; and An upstream inner primer having a nucleotide sequence as shown in SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124 or SEQ ID NO: 128; and A downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 111, SEQ ID NO: 117, SEQ ID NO: 121, SEQ ID NO: 125 or SEQ ID NO: 129; and An upstream loop primer having a nucleotide sequence as shown in SEQ ID NO: 112 or SEQ ID NO: 130; and / or A downstream loop primer having a nucleotide sequence as shown in SEQ ID NO: 113 or SEQ ID NO: 131; The detection primer set of the Bacillus cereus comprises: The upstream external primers with nucleotide sequences as shown in SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144 or SEQ ID NO: 148, and A downstream external primer with a nucleotide sequence as shown in SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145 or SEQ ID NO: 149, and The upstream inner primer has a nucleotide sequence as shown in SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146 or SEQ ID NO: 150, and The nucleotide sequence of the downstream internal primer is shown as SEQ ID NO:135, SEQ ID NO:139, SEQ ID NO:143, SEQ ID NO:147 or SEQ ID NO:

151.

2. The primer set according to claim 1, characterized in that The detection primer set of Staphylococcus epidermidis includes: An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 2, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 3, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 4; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO:5, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:6, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO:7, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:8; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO:9, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:10, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO:11, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:12; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 13, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 14, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 15, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 16; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 17, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 18, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 19, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 20; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 21, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 22, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 23, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 24; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 25, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 26, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 27, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 28; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 29, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 30, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 31, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 32; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 33, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 34, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 35, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 36; and / or An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO:37, a downstream outer primer having a nucleotide sequence as shown in SEQ ID NO:38, an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:39, and a downstream inner primer having a nucleotide sequence as shown in SEQ ID NO:40; The detection primer set of Pseudomonas aeruginosa comprises: An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO:41, a downstream outer primer having a nucleotide sequence as shown in SEQ ID NO:42, an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:43, a downstream inner primer having a nucleotide sequence as shown in SEQ ID NO:44, an upstream loop primer having a nucleotide sequence as shown in SEQ ID NO:45, and a downstream loop primer having a nucleotide sequence as shown in SEQ ID NO:46; and / or an upstream external primer having a nucleotide sequence as shown in SEQ ID NO:47, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:48, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO:49, a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:50, an upstream loop primer having a nucleotide sequence as shown in SEQ ID NO:51, and a downstream loop primer having a nucleotide sequence as shown in SEQ ID NO:52; and / or An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO:53, a downstream outer primer having a nucleotide sequence as shown in SEQ ID NO:54, an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:55, a downstream inner primer having a nucleotide sequence as shown in SEQ ID NO:56, and an upstream loop primer having a nucleotide sequence as shown in SEQ ID NO:57; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO:58, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:59, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO:60, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:61; and / or an upstream external primer having a nucleotide sequence as shown in SEQ ID NO:62, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:63, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO:64, a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:65, and an upstream loop primer having a nucleotide sequence as shown in SEQ ID NO:66; The detection primer set of Staphylococcus aureus comprises: An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 67, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 68, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 69, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 70; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 71, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 72, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 73, a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 74, and a downstream loop primer having a nucleotide sequence as shown in SEQ ID NO: 75; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 76, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 77, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 78, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 79; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 80, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 81, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 82, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 83; and / or An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO:84, a downstream outer primer having a nucleotide sequence as shown in SEQ ID NO:85, an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO:86, and a downstream inner primer having a nucleotide sequence as shown in SEQ ID NO:87; The detection primer set of the hemolytic streptococcus comprises: An upstream external primer having a nucleotide sequence as shown in SEQ ID NO:88, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:89, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO:90, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:91; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO:92, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO:93, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO:94, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO:95; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 96, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 97, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 98, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 99; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 100, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 101, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 102, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 103; and / or An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO: 104, a downstream outer primer having a nucleotide sequence as shown in SEQ ID NO: 105, an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO: 106, and a downstream inner primer having a nucleotide sequence as shown in SEQ ID NO: 107; The detection primer set of Streptococcus pneumoniae includes: An upstream outer primer having a nucleotide sequence as shown in SEQ ID NO: 108, a downstream outer primer having a nucleotide sequence as shown in SEQ ID NO: 109, an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO: 110, a downstream inner primer having a nucleotide sequence as shown in SEQ ID NO: 111, an upstream loop primer having a nucleotide sequence as shown in SEQ ID NO: 112, and a downstream loop primer having a nucleotide sequence as shown in SEQ ID NO: 113; and / or an upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 114, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 115, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 116, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 117; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 118, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 119, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 120, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 121; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 122, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 123, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 124, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 125; and / or an upstream outer primer having a nucleotide sequence as shown in SEQ ID NO: 126, a downstream outer primer having a nucleotide sequence as shown in SEQ ID NO: 127, an upstream inner primer having a nucleotide sequence as shown in SEQ ID NO: 128, a downstream inner primer having a nucleotide sequence as shown in SEQ ID NO: 129, an upstream loop primer having a nucleotide sequence as shown in SEQ ID NO: 130, and a downstream loop primer having a nucleotide sequence as shown in SEQ ID NO: 131; The detection primer set of the Bacillus cereus comprises: An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 132, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 133, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 134, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 135; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 136, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 137, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 138, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 139; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 140, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 141, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 142, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 143; and / or An upstream external primer having a nucleotide sequence as shown in SEQ ID NO: 144, a downstream external primer having a nucleotide sequence as shown in SEQ ID NO: 145, an upstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 146, and a downstream internal primer having a nucleotide sequence as shown in SEQ ID NO: 147; and / or The nucleotide sequence is an upstream external primer as shown in SEQ ID NO:148, the nucleotide sequence is a downstream external primer as shown in SEQ ID NO:149, the nucleotide sequence is an upstream internal primer as shown in SEQ ID NO:150, and the nucleotide sequence is a downstream internal primer as shown in SEQ ID NO:

151.

3. A detection reagent, characterized in that: The method comprises a detection mixture and a primer set according to claim 1 or 2; the detection mixture comprises Bst DNA polymerase, dNTP, betaine, magnesium sulfate, Bst LF Buffer with Mg 2+ and / or at least one of an indicator reagent.

4. The detection reagent according to claim 3, characterized in that The indicator reagent is selected from SYBR Green I and / or calcein.

5. The detection reagent according to claim 3 or 4, characterized in that The detection mixture includes: 0.15 μM to 0.4 μM Bst DNA polymerase, 0.2 mM to 10 mM dNTP, 0 M to 1.2 M betaine, 2 mM to 10 mM magnesium sulfate, 1× Bst LF Buffer with Mg 2+ , 1-2 mM calcein.

6. A kit, characterized in that Including at least one of positive quality control products and / or negative quality control products and at least one of the following i) to ii): i), the primer set according to claim 1 or 2; ii) The detection reagent according to any one of claims 3 to 5.

7. A method for detecting bacteria in intraocular fluid for non-diagnostic purposes, characterized in that: The sample is tested using at least one of the following I) to III): 1), the primer set according to claim 1 or 2; II), the detection reagent according to any one of claims 3 to 5; III), the kit according to claim 6.

8. The detection method according to claim 7, characterized in that: The method comprises the following steps: mixing the detection reagent according to any one of claims 3 to 5 with a sample to be tested and reacting the mixture, and judging whether the sample is infected with pathogenic bacteria according to the reaction result.

9. The detection method according to claim 9, characterized in that: The judgment standard is: if the CT value is less than 35 and there is a standard S curve, it is positive, otherwise it is negative.

10. The detection method according to any one of claims 7 to 9, characterized in that: The samples include conjunctival sac secretions, tears, aqueous humor and / or vitreous humor.

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