Mg is cultivated in 1. 1 medium, which requires extremely high culture medium and grows slowly, so it is difficult to cultivate Mg, especially in clinical specimens. In Tully 198 1 year, SP-4 culture medium which is of great significance to medical mycoplasma was established. SP-4 medium suitable for Mg growth does not contain thallium acetate, glucose and other nutrients needed for Mg metabolism. The optimum pH is 7.4-7.5, and it can be solidified by adding 0.8% noble agar or 0.5-0.6% agar for strain cloning and colony observation. Tully et al. used this medium to isolate and culture two Mg strains from 13 urethral secretion samples, and the culture period was about 50 days. Jensen and others also reported another one. That is, the improved Friis broth medium, 1 1 copy of PCR detection of Mg-DNA positive urethral secretion samples, of which 6 were positive growth.

Domestic scholars have improved SP-4 culture medium. Zhao Jiwen and others [4] successfully isolated Mg from sexually transmitted diseases and promiscuous people by using improved SP-4 medium. The initial growth time is more than 30 days, with an average of 37.83 days and the longest of 55 days.

1.2 tissue cell culture method can provide a good growth environment similar to that in vivo for mycoplasma. As early as 1960s, Chanock et al. [5] reported the growth of Mycoplasma pneumoniae (Mp) in tissues and cells. In 1990s, Jensen et al. [3,6] began to try to proliferate Mg by cell culture, and observed the process of Mg invading cells and its location in cells under electron microscope. Under the monitoring of PCR, Jensec found that among the urethral samples with positive MgDNA detected by 1 1 PCR, 9 samples were adapted to proliferate in Vero cell magnetic head. After several passages, they were transferred to the improved Friis fF broth medium, and 6 samples could continue to be passaged. Finally, 4 strains were cloned in agar medium. Jensen believes that the process of cell culture and reproduction has played three roles in isolating and obtaining new Mg strains. Firstly, Mg in clinical specimens should be gradually adapted to grow in artificial culture medium; secondly, the number of mycoplasma inoculated in mycoplasma broth culture medium should be increased; thirdly, a continuous inoculation source should be provided. According to the immunological characteristics of Mg, a serological method for detecting antibody and antigen of Mg was established.

Furr et al. [7] reported MIF technique for detecting Mg antibody in detail in 1984. Moller et al used MIF to detect Mg antibody in 3 1 patients with acute pelvic inflammatory disease, and found that about 40% of them had four or more changes in antibody titer within one month after onset. Taylor-Robinson et al [9] reported that indirect MIF test is a sensitive, specific and simple method to detect Mg antibody in patients with NGU. Jensen et al. used EIA to detect Mg antibody in acute blood samples of male STD patients with urethral inflammation and without urethritis symptoms, and found that the reactivity was weak and there was a wide cross-reaction with Mp.

According to the characteristics that specific antibodies can prevent the growth and metabolism of mycoplasma, we can use known high-priced blood to carry out ancestor growth and metabolism inhibition test to identify mycoplasma [1 1]. Zhao Jiwen and others identified the isolated Mg strain by MIT.

Lipid-associated membrane proteins (LAMPs) exposed on the surface of mycoplasma are species-specific antigens with high antigenicity. LAMP antibodies of different strains of mycoplasma are highly species-specific and have no cross-reaction with other species [12, 13]. ) Therefore, LAMp extracted from Mg established an ELISA method to detect Mg antibody, which can eliminate the cross reaction between Mg and Mp. Detection of Mg by DNA probe and polymerase chain reaction (PCR) overcomes the shortcomings of the first two detection methods, and provides a powerful means for studying the relationship between Mg and disease etiology.

3. 1DNA probe technology 1987 Hyman[ 14] The library of Mg gene was constructed with PUCB vector, and specific DNA probes were screened from it. The specific mycoplasma DNA containing only 0. 1ng, namely 105CFU, can be detected by dot blot hybridization.

3.2 Polymerase chain reaction (PCR) technology PCR is a new gene diagnosis technology with high sensitivity, strong specificity, rapidity and simplicity. From 65438 to 0990, PCR technology began to be applied to medical branches.

The early primers were designed according to the DNA sequence of bacterial terminal specific adhesion protein. Palmer et al. amplified part of the nucleotide sequence of Mg adhesion protein in vitro, and all three strains of Mg showed 37bp DNA fragments, which proved that 10- 15g of mg-DNA could be detected by PCR, and the primer sequence was as follows:

mg 1:5 ' TGT CTA TGACCA GTA TGT AC3 '

Mg2:5“CTG CTT TGG TCA AGA CAT CA3”

199 1 year Jensen et al. [16] designed and synthesized the following groups of primers according to the adhesion protein gene of Mg:

mgPa- 1 5 ' AGA TGA TGA AAC CCT AAC CCC TTG G3 '

MgPa- 1 5'GAC Caterpillar CAA GGT ATT TCT CAA CAG C3'

mgPa- 1 5 ' CCG AGG GGT TTT CCA TTT TTG C3 '

MgPa- 1 and MgPa-3 successfully amplified the 28 1bpDNA fragment of Mg, and five clinical isolates amplified the same DNA fragment, but all other mycoplasma and bacteria were negative. Using MgPa-2 as a probe, the amplification products of Southern eP blot hybridization were all positive, which proved that the primers were specific, and the detection of * * * reduced about 50 Mg cells. 1993 Jensen et al. [17] designed another pair of primers according to the Mg mucosal protein gene, namely:

Another pair of primers was designed for mg adhesion protein gene, namely:

mgPa-476:5 ' ATG GCG AGC CTA TCT TTG ATC CTT TAA 3 '

mgPa-903:5 ' TTC ACC TCC CCA CTA CTG TCC TAA tgc 3 '

It is used to confirm and supplement the amplification results of primers MgPa- 1 and MgPa-3, and the amplified fragment is 453bp. It was found that the sensitivity of these two pairs of Mg adhesion protein primers was exactly the same. Urine swabs of 99 patients with urethritis were detected by this method, and the results showed that 17 cases were Mg positive, but none of them were positive by culture method.

1994 Jensen[ 18] amplified the gene sequence of Mg adhesion protein and sequenced it. The results show that the sequence under Mg condition has obvious heterogeneity. Therefore, according to the gene sequence of 16SrRNA, Jensen and other scholars designed specific PCR primers to detect all Mg infections in clinical samples.

mg- 1.5 ' GAA TGA CTC TAG CAG GCA ATG GCT G3 '

Mg-2，5 ' ATT TGC TGC TCA CTT TTA CAA GTT ggct 3 '

The experimental results of four clinical specimens show that the gene sequence of Mg adhesion protein is different, but its 165rRN gene sequence 100% is homologous. By combining the two primers for PCR, the Mg gene with less than 10-50 copies can be detected in suspected positive samples, which ensures the PCR system with 16SrRNA gene as the target gene.

Zhao Jiwen et al [19] used the primers designed by Palmer to amplify the DNA fragment of mg 37 BP, and Mg was detected in three populations. The results showed that the positive rate of promiscuous people was 25.26%, the positive rate of sexually transmitted diseases was 65438 02.33%, and the positive rate of healthy people was 3.85%. Kong et al [20] used MgPa- 1 and MgPa-3 designed by Jensen to amplify Mg28 1bpDNA fragment. The results showed that the detection rate of Mg in promiscuous women was 65438 00.2%, and that in sexually transmitted diseases patients was 4.0%.

In 1998, Mi Huangzu et al. [2] reported two nested PCR detection techniques based on Mg adhesion protein and 16rRNA gene respectively. The former amplified 374bpDNA fragment of original Mg, while the latter amplified 24 1bpDNA fragment. Nested PCR not only improves the sensitivity, but also further improves the characteristics because of the use of two different pairs of primers. 40 female patients with sexually transmitted diseases were detected by these methods, and 12 cases were positive, with a positive rate of 30%, which was higher than that of ordinary PCR. The "gold standard" of microbial infection diagnosis is culture method, but the success rate of Mg culture is extremely low, which needs further research and improvement to promote the growth of Mg in it. In addition, it may be a good method to introduce cell lines into the culture of Mg, so it is necessary to strengthen the exploration and research in this field. Because of the cross reaction between mycoplasma species and the high purity of antigen and antibody, the immunological detection method is limited in practical application. It is of great practical value to study and establish a rapid, sensitive and specific method for detecting Mg antigen in clinical specimens. PCR is a simple and direct method to detect Mg, which is being widely used. It is mainly used for early diagnosis of Mg infection or acute infection and epidemiological study of Mg in various populations. However, it should be noted that: ① low Mg content, insufficient DNA purity, too many inhibitors and low TaqDNA polymerase activity in clinical samples will all cause false negatives, and the extraction technology of template DNA needs to be further improved; ② Due to the high sensitivity of PCR, the contamination of PCR products should be strictly avoided in the whole operation process to reduce false positives.